Herpesvirus with Modified Glycoprotein B

ABSTRACT

The present invention is directed to a recombinant herpesvirus comprising a heterologous polypeptide ligand capable of binding to a target molecule and fused to or inserted into glycoprotein B at specific sites. The herpesvirus may comprise more than one ligand, and the additional ligand(s) may be comprised by a modified glycoprotein D and/or modified glycoprotein H. This allows the herpesvirus to target a cell for therapeutic purposes, and a cell for virus production. The present invention further comprises a pharmaceutical composition comprising the herpesvirus, the herpesvirus for use in the treatment of a tumor, infection, degenerative disorder or senescence-associated disease, a nucleic acid and a vector coding for the gB, a polypeptide comprising the gB, and a cell comprising the herpesvirus, nucleic acid, vector or polypeptide. Moreover, a method for infecting a cell with the herpesvirus or for producing the herpesvirus is disclosed.

BACKGROUND OF THE INVENTION

The work leading to this invention has received funding from theEuropean Research Council under the European Union's Seventh FrameworkProgram (FP7/2007-2013)/ERC grant agreement n° 340060.

Despite a steady development in healthcare, the burden of diseases andpathologies that cannot be treated or cannot be sufficiently treated,remains elevated. Eminent among these are numerous forms of tumors, inparticular metastatic forms of tumors that are treated withchemo-radio-therapy or biological medicaments, or combinations thereof,however, with limited success.

An alternative approach of tumor treatment is oncolytic virotherapy,whereby a replication competent virus infects the tumor cells, spreadsfrom cell to cell of the tumor and destroys them.

Oncolytic virotherapy can be combined with immunotherapy of cancers.Thus the patient may be administered both oncolytic virus and theimmunotherapeutic agents, or, the oncolytic virus has been engineered toexpress a cytokine, chemokine, or molecules such as immune checkpointregulators, that boost the immune response of the host to the tumor.Immune checkpoint regulators include antibodies or single chainantibodies to CTLA4, PD1, PDL1, LAG3, KIR, NKG2A, TIM3, TIGIT, CD96,BTLA. They can be administered singly or in combination. Included inthis category of recombinant oncolytic viruses capable to elicit animmune response to the tumor is T-VEC, renamed Talimogene laherparepvec(commercial name Imlygic), an HSV that encodes GM-CSF, approved by FDAfor the treatment of metastatic melanoma

Herpes simplex virus (HSV) is a pathogen virus for humans. In culture,it infects a large number of mammalian cells. It is an enveloped viruswhich enters the cell by membrane fusion, either at the plasma membraneor through endocytosis, depending on the target cell type. Entry of HSVinto a target cell is a multistep process, requiring complexinteractions and conformational changes of viral glycoproteins gD,gH/gL, gC and gB. These glycoproteins constitute the virus envelopewhich is the most external structure of the HSV particle and consists ofa membrane. For cell entry, gC and gB mediate the first attachment ofthe HSV particle to cell surface heparan sulphate. Thereafter, gD bindsto at least two alternative cellular receptors, being Nectin-1 and HVEMor HVEA, causing conformational changes in gD that initiates a cascadeof events leading to virion-cell membrane fusion. Thereby, theintermediate protein gH/gL (a heterodimer) is activated which triggersgB to catalyze membrane fusion. Thereby, gB is membrane bound andfunctions as a viral fusogen.

Oncolytic HSVs (o-HSV) have been used in recent years as oncolyticagents. As wild-type HSV viruses are highly virulent, there is arequirement that the o-HSVs are attenuated. T-VEC/Imlygic and theviruses that have reached clinical trials carry deletion of one or moreHSV genes, including the gamma γ₁34.5 gene, which encodes the ICP34.5protein whose role is to preclude the shut off of protein synthesis ininfected cells, and the UL39 gene, which encodes the large subunit ofribonucleotide reductase. In addition to some disadvantages which areshown by these viruses, such as the failure to produce high yield ofprogeny viruses, they furthermore have the preserved ability to bind toany cell bearing their natural receptors. Thus, the therapeutic effectof tumor cell killing is diminished and the viruses may have limitationsin medical use.

One approach to overcome these limits has been genetic engineering ofo-HSVs which exhibit a highly specific tropism for the tumor cells, andare otherwise not attenuated. This approach has been defined asretargeting of HSV tropism to tumor-specific receptors.

The retargeting of HSV to cancer-specific receptors entails the geneticmodifications of gD, such that it harbors heterologous sequences whichencode a specific ligand. Upon infection with the recombinant virus,progeny viruses are formed which carry in their envelope the chimericgD-ligand glycoprotein, in place of wildtype gD. The ligand interactswith a molecule specifically expressed on the selected cell and enablesentry of the recombinant o-HSV into the selected cell. Examples ofligands that have been successfully used for retargeting of HSV areIL13a, uPaR, a single chain antibody to HER2 and a single chain antibodyto EGFR.

The retargeting through modification of glycoproteins has also beenattempted with gC. The inserted ligands were EPO and IL13. The viruscarrying the gC-EPO polypeptide attached to cells expressing the EPOreceptor. However, this attachment did not lead to infectious entry. Inaddition, the gC-IL13 polypeptide was present in a virus that carried asecond copy of IL13 in the gD gene. Therefore, it cannot be inferredfrom those studies whether the gC-IL13 contributed or not to theretargeting to the IL13 alpha2 receptor.

The retargeting through genetic modification of gH has also beenachieved. The inserted ligand was a single-chain antibody (scFv)directed to HER2, without or with deletions within the gH gene. Thevirus was successfully retargeted to a cell carrying the HER2 receptor(Gatta et al., 2015). In addition, a recombinant virus was constructedwhich contained the scFv directed to HER2 in gH and an scFv directed toEGFR in the mature gD protein. This resulted in double retargeting tothe cell carrying the receptors. Further, a recombinant virus wasconstructed which contained the scFv directed to HER2 in gH and the scFvdirected to HER2 in the mature gD protein. This resulted in doubleretargeting to the HER2 receptors (PCT application (Abstract # P-28,9^(th) International conference on Oncolytic virus Therapeutics, Boston2015).

The retargeting of viruses via gB has never been reported. Whileinsertion sites within gB gene were identified which resulted in viableviral mutants with preserved membrane fusion activity (Gallagher et al.,2014; Lin and Spear, 2007; Potel et al., 2002), the assays used foranalyzing the polypeptide-gB fusions did not predict whether—in the casethat the inserted polypeptide is a heterologous peptide capable to binda target receptor—the ensuing recombinant would contribute to the fusionactivity of gB and exhibit a tropism re-addressed (retargeted) to thereceptor targeted by the ligand. Foremost, the experience in the artpredicts that any effort of retargeting the tropism of a virus,including HSV, is only successful if the glycoprotein chosen formodifications, e.g. for insertion of a heterologous ligand, is adeterminant of the virus tropism. Receptors have been claimed for HSVgB; they are heparan sulfate proteoglycans to which gB and gC bind,Myelin-associated glycoprotein MAG, paired immunoglobulin-like type 2receptor alpha (PILRalpha), DC-SIGN and non-muscle myosin heavy chain 9MYH9/NMHC-IIA. In no case, the interaction of gB with these moleculeswas shown to determine

HSV tropism. Thus, PILRalpha participates in entry of HSV into monocyticcells, a cell type not usually targeted by HSV, a virus which infectspreferentially epidermal and neuronal cells. For the other receptors therole they play in HSV infection was not investigated. Hence, an expertin the art can not predict that suitable modifications to gB can resultin retargeted tropism to the target receptor of choice.

There is a need in the art to provide several alternative retargetingstrategies. This need stems from the heterogeneity of cancer cells in asame tumor, whereby cells express different receptors, the need toeliminate cancer stem cells, which may express a repertoire of receptorsdifferent from those of the cancer cells, or the insurgence of cellsresistant to targeted therapy.

The present invention describes a recombinant HSV with a modified gBprotein which retargets the virus to receptors of cells which need to beeliminated.

The present inventors have shown that it is possible to construct arecombinant HSV which comprises a polypeptide ligand directed to aspecific cellular receptor as a fusion protein with gB, whereby due tothe presence of the ligand, the HSV is retargeted to cells carrying thereceptor. Furthermore, the HSV has been shown to maintain infectivity,resulting in the entry into the cells carrying the receptor and killingof the infected cells.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the present invention is described in detail. Thefeatures of the present invention are described in individualparagraphs. This, however, does not mean that a feature described in aparagraph stands isolated from a feature or features described in otherparagraphs. Rather, a feature described in a paragraph can be combinedwith a feature or features described in other paragraphs.

The term “comprise/es/ing”, as used herein, is meant to “include orencompass” the disclosed features and further features which are notspecifically mentioned. The term “comprise/es/ing” is also meant in thesense of “consist/s/ing of” the indicated features, thus not includingfurther features except the indicated features. Thus, the product of thepresent invention may be characterized by additional features inaddition to the features as indicated.

In a first aspect, the present invention provides a recombinantherpesvirus comprising a heterologous polypeptide ligand capable ofbinding to a target molecule and fused to or inserted into glycoproteinB (gB) present in the envelope of the herpesvirus, wherein the ligand isfused to gB, or wherein the ligand is inserted at any amino acid withina disordered region of gB, but is not inserted at any amino acid withinthe region spanning from amino acids 77 to 88 of gB according to SEQ IDNO: 1 or within a corresponding region of a homologous gB, or whereinthe ligand is inserted at any amino acid within a region spanning fromamino acids 31 to 77 or 88 to 184, preferably amino acids 31 to 77 or 88to 136 or more preferably 31 to 77 or 88 to 108, and/or within a regionspanning from amino acids 409 to 545, preferably amino acids 459 to 545or more preferably amino acids 459 to 497, or still more preferablyamino acids 460 to 491, of gB according to SEQ ID NO: 1 or within acorresponding region of a homologous gB.

Furthermore, the present invention provides a recombinant herpesviruscomprising a heterologous polypeptide ligand capable of binding to atarget molecule and inserted into glycoprotein B (gB) present in theenvelope of the herpesvirus, wherein the ligand has a length of 5 to 120amino acids and is inserted at any amino acid within a region spanningfrom amino acids 77 to 88 of gB according to SEQ ID NO: 1 or within acorresponding region of a homologous gB.

In an embodiment thereof, the herpesvirus has the capability of bindingto a cell expressing or binding the target molecule, preferably offusing with the cell, more preferably of entering the cell, mostpreferably of killing the cell.

In an embodiment thereof, the target molecule is present on a diseasedcell, preferably the diseased cell is a tumor cell, an infected cell, adegenerative disorder-associated cell or a senescent cell, or the targetmolecule is present on a cell present in cell culture, preferably thecell is a cultured cell suitable for growth of the herpesvirus, morepreferably a cell line approved for herpesvirus growth, even morepreferably a Vero, 293, 293T, HEp-2, HeLa, BHK, or RS cell, mostpreferably a Vero cell.

In an embodiment thereof, the target molecule present on a diseased cellis a tumor-associated receptor, preferably a member of the EGF receptorfamily, including HER2, EGFR, EGFRIII, or EGFR3 (ERBB3), EGFRvIII, orMET, FAP, PSMA, CXCR4, CEA, CADC, Mucins, Folate-binding protein, GD2,VEGF receptors 1 and 2, CD20, CD30, CD33, CD52, CD55, the integrinfamily, IGF1R, the Ephrin receptor family, the protein-tyrosine kinase(TK) family, RANKL, TRAILR1, TRAILR2, IL13Ralpha, UPAR, Tenascin, amember of the immune checkpoint family regulators, including PD-1,PD-L1, CTL-A4, TIM-3, LAG3, or IDO, tumor-associated glycoprotein 72,ganglioside GM2, A33, Lewis Y antigen, or MUC1, most preferably HER2, orthe target molecule present on a cell present in cell culture is anartificial molecule, preferably an antibody, an antibody derivative oran antibody mimetic, more preferably a single-chain antibody (scFv),still more preferably an scFv capable of binding to a part of the GCN4yeast transcription factor, still more preferably an scFv capable ofbinding to the part of the GCN4 yeast transcription factor as comprisedby SEQ ID NO: 37, still more preferably the scFv as comprised by SEQ IDNO: 39, most preferably the molecule identified by the sequence of SEQID NO: 41.

In an embodiment thereof, the ligand is a natural polypeptide or anartificial polypeptide, preferably the ligand is capable of binding to atarget molecule present on a cell present in cell culture or to a targetmolecule present on a diseased cell, more preferably the ligand is anatural ligand of a target molecule which is accessible on a cell, apart of the natural ligand capable of binding to the target molecule, apart of a natural polypeptide, an antibody, an antibody derivative, anantibody mimetic, still more preferably the ligand is a part of thenatural polypeptide capable of binding to a target molecule present on acell present in cell culture or an scFv, still more preferably theligand is a part of the GCN4 yeast transcription factor such as the partof the GCN4 yeast transcription factor as comprised by SEQ ID NO: 37 oran scFv capable of binding to a target molecule present on a tumor cell,preferably HER2, most preferably the ligand is the molecule identifiedby the sequence of SEQ ID NO: 37 or the scFv identified by SEQ ID NO:32.

In an embodiment thereof, the target molecule is HER2, the ligand is anscFv as identified by SEQ ID NO: 32 and the diseased cell is a tumorcell expressing HER2, preferably a breast cancer cell, ovary cancercell, stomach cancer cell, lung cancer cell, head and neck cancer cell,osteosarcoma cell, glioblastoma multiforme cell, or salivary gland tumorcell, and/or the target molecule is the molecule identified by thesequence of SEQ ID NO: 41, the ligand is the molecule identified by thesequence of SEQ ID NO: 37, and the cell is present in cell culture andexpresses the molecule identified by the sequence of SEQ ID NO: 41.

In an embodiment thereof, one or more ligands are fused to or insertedinto gB, preferably the gB comprises a ligand capable of binding to atarget molecule present on a cell present in cell culture and a ligandcapable of binding to a target molecule present on a diseased cell.

In an embodiment thereof, the herpesvirus comprises a modified gD and/ora modified gH, preferably wherein the gB comprises a ligand capable ofbinding to a target molecule present on a cell present in cell cultureand the modified gD and/or the modified gH comprise(s) a ligand capableof binding to a target molecule present on a diseased cell, mostpreferably the gB comprises the sequence identified by SEQ ID NO: 37,the target molecule is the molecule with the sequence identified by SEQID NO: 41, and the cell is present in cell culture and expresses themolecule identified by the sequence of SEQ ID NO: 41, and the modifiedgD and/or the modified gH comprise(s) an scFv identified by SEQ ID NO:32, the target molecule is HER2, and the cell is a tumor cell expressingHER2, preferably a breast cancer cell, ovary cancer cell, stomach cancercell, lung cancer cell, head and neck cancer cell, osteosarcoma cell,glioblastoma multiforme cell, or salivary gland tumor cell.

In an embodiment thereof, the gD is modified to have a deletion of aminoacids 30 to 38 of gD or a subset thereof, preferably the gD is modifiedto have a deletion of amino acid 30 and/or amino acid 38, morepreferably the gD is modified to have a deletion of amino acid 30 andamino acid 38, with regard to mature gD according to SEQ ID NO: 62 or acorresponding region of a homologous gD.

In an embodiment thereof, a heterologous polypeptide ligand is insertedinto gD instead of amino acids 30 to 38 or a subset thereof, preferablythe heterologous polypeptide ligand is inserted instead of amino acid 30or amino acid 38, more preferably the heterologous polypeptide ligand isinserted instead of amino acid 38 and amino acid 30 is deleted, withregard to mature gD according to SEQ ID NO: 62 or a corresponding regionof a homologous gD.

In an embodiment thereof, the herpesvirus encodes one or moremolecule(s) that stimulate(s) the host immune response against a cell,preferably a diseased cell.

Glycoprotein B (gB) is an envelope protein which is present on the outersurface of herpesviridae and is involved in the binding of the virus toa cell and invasion into the cell. Among the glycoproteins which areinvolved in cell entry, gB is the fusogen that undergoesfusion-promoting conformational rearrangement upon stimulation via gDand gH/gL. gB is composed of 904 amino acids including 30 amino acidssignal peptide, 696 amino acids ectodomain, 69 amino acids transmembranedomain, and 109 amino acids C-tail. gB belongs to the most highlyconserved glycoproteins across the Herpesviridae family. The crystalstructure of herpes simplex virus (HSV) type 1 gB was solved in itspost-fusion conformation; it is a trimer, with five structural domains(I-V). Domain I extends from amino acids 154 to 363, domain II extendsfrom amino acids 142 to 153 and 364 to 459, followed by the disorderedregion of amino acids 460 to 491, domain III extends from amino acids117 to 133, 500 to 572, and 661 to 669, domain IV extends from aminoacids 111 to 116 and 573 to 660, and domain V extends from amino acids670 to 725 (Heldwein et al., 2006). The N-terminal region with itsdisordered structure extends from amino acids 31 to 108. The crystalstructures of EBV and HCMV were also solved, and are essentially similarto that of HSV type 1 (Backovic et al., 2009; Burke and Heldwein, 2015).Due to its unique structure, herpesvirus gBs belong to a new class ofviral membrane fusion glycoproteins, class III. The nuciceotide andamino acid sequences of a variety of gBs of different herpesviruses areknown in the art. For illustrative purposes only, without being limitedthereto, reference is made to the amino acid sequence of gB of humanherpesvirus 1 disclosed herein as SEQ ID NO: 1. The correspondingnucleotide sequence and the amino acid sequence are available from theNCBI (National

Centre for Biotechnology Information; National Library of Medicine,Bethesda, Md. 20894, USA; www.ncbi.nlm.nih.gov) under the accessionnumber “Genome”, GU734771.1, coordinates from 52996 to 55710.

SEQ ID NO: 1   1MRQGAPARGC RWFVVWALLG LTLGVLVASA APSSPGTPGV AAATQAANGG PATPAPPAPG  61PAPTGDTKPK KNKKPKNPPP PRPAGDNATV AAGHATLREH LRDIKAENTD ANFYVCPPPT 121GATVVQFEQP RRCPTRPEGQ NYTEGIAVVF KENIAPYKFK ATMYYKDVTV SQVWFGHRYS 181QFMGIFEDRA PVPFEEVIDK INAKGVCRST AKYVRNNLET TAFHRDDHET DMELKPANAA 241TRTSRGWHTT DLKYNPSRVE AFHRYGTTVN CIVEEVDARS VYPYDEFVLA TGDFVYMSPF 301YGYREGSHTE HTSYAADRFK QVDGFYARDL TTKARATAPT TRNLLTTPKF TVAWDWVPKR 361PSVCTMTKWQ EVDEMLRSEY GGSFRFSSDA ISTTFTTNLT EYPLSRVDLG DCIGKDARDA 421MDRIFARRYN ATHIKVGQPQ YYLANGGFLI AYQPLLSNTL AELYVREHLR EQSRKPPNPT 481PPPPGASANA SVERIKTTSS IEFARLQFTY NHIQRHVNDM LGRVAIAWCE LQNHELTLWN 541EARKLNPNAI ASATVGRRVS ARMLGDVMAV STCVPVAADN VIVQNSMRIS SRPGACYSRP 601LVSFRYEDQG PLVEGQLGEN NELRLTRDAI EPCTVGHRRY FTFGGGYVYF EEYAYSHQLS 661RADITTVSTF IDLNITMLED HEFVPLEVYT RHEIKDSGLL DYTEVQRRNQ LHDLRFADID 721TVIHADANAA MFAGLGAFFE GMGDLGRAVG KVVMGIVGGV VSAVSGVSSF MSNPFGALAV 781GLLVLAGLAA AFFAFRYVMR LQSNPMKALY PLTTKELKNP TNPDASGEGE EGGDFDEAKL 841AEAREMIRYM ALVSAMERTE HKAKKKGTSA LLSAKVTDMV MRKRRNTNYT QVPNKDGDAD 901EDDL

gB homologs are found in all members of the Herpesviridae. Therefore,the term “glycoprotein B”, as referred to herein, refers to any gBhomolog found in Herpesviridae. Alternatively, gB, as referred toherein, refers to any gB which has an amino acid identity to thesequence of SEQ ID NO: 1 of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100%. Alternatively, the gB, as referred to herein, refersto any gB which has an amino acid homology to SEQ ID NO: 1 of at least50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%. The gB, as referred toherein, also includes a fragment of gB. Preferably, gB, as referred toherein, including any gB found in Herpesviridae, any gB having an aminoacid identity to the sequence of SEQ ID NO: 1, as defined above, and anyfragment of a gB, has the same activity of the gB according to SEQ IDNO: 1. More preferably, during the entry process of the virus into acell, gB undergoes a conformational change promoting fusion of the viruswith the membrane of the cell, and still more preferably, it acts as afusogen mediating the fusion of the virus with the cell membrane.

The percentage of “sequence identity,” as used herein, refers to thepercentage of amino acid residues which are identical in correspondingpositions in two optimally aligned sequences. It is determined bycomparing two optimally aligned sequences over a comparison window,where the fragment of the amino acid sequence in the comparison windowmay comprise additions or deletions (e.g., gaps or overhangs) ascompared to the reference sequence, SEQ ID NO: 1 (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of

Smith and Waterman, 1981, by the homology alignment algorithm ofNeedleman and Wunsch, 1970, by the search for similarity method ofPearson and Lipman, 1988, by the algorithm of Karlin and Altschul, 1990,modified by Karlin and Altschul, 1993, or by computerizedimplementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, andTFASTA in the Wisconsin Genetics Software

Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection. GAP and BESTFIT are preferably employed to determinethe optimal alignment. Typically, the default values of 5.00 for gapweight and 0.30 for gap weight length are used.

The “percentage of homology”, as used herein, refers to the percentageof amino acid residues which are homologous in corresponding positionsin two optimally aligned sequences. The “percentage of homology” betweentwo sequences is established in a manner substantially identical to whathas been described above with reference to the determination of the“percentage of identity” except for the fact that in the calculationalso homologous positions and not only identical positions areconsidered. Two homologous amino acids have two identical or homologousamino acids. Homologous amino acid residues have similarchemical-physical properties, for example, amino acids belonging to asame group: aromatic (Phe, Trp, Tyr), acid (Glu, Asp), polar (Gln, Asn),basic (Lys, Arg, His), aliphatic (Ala, Leu, lie, Val), with a hydroxylgroup (Ser, Thr), or with a short lateral chain (Gly, Ala, Ser, Thr,Met). It is expected that substitutions between such homologous aminoacids do not change a protein phenotype (conservative substitutions).

A gB is “homologous” or a “homolog” if it has an identity to SEQ ID NO:1 of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, ifit has an amino acid homology to SEQ ID NO: 1 of at least 50%, 60%, 70%,80%, 85%, 90%, 95%, or 100%, or if it has the same activity as the gBaccording to SEQ ID NO: 1. Preferably, “same activity” may be understoodin the sense that gB binds to a cellular receptor, and more preferably,during the entry process of the virus into a cell, gB undergoes aconformational change promoting fusion of the virus with the membrane ofthe cell, and still more preferably, it acts as a fusogen. A homolog mayalso be a fragment of a full length gB having the activity as indicatedabove.

The chimeric gB of the present invention (as exemplified by SEQ ID NO:2) carries a heterologous polypeptide ligand and thereby confers a newactivity on the virus, in addition to the activity that the gB portioncarries out for the wildtype (wt) virus. The chimeric gB, once it ispart of the envelope of the recombinant virus, enables the binding ofthe recombinant virus to the target molecule, and retargets the tropismof recombinant virus to a cell carrying the target molecule of theligand.

Preferably, the chimeric gB undergoes a conformational change promotingfusion of the virus with the membrane of the cell, and still morepreferably, the chimeric gB acts as a fusogen. After fusion with a cellcarrying the target molecule of the ligand, the recombinant herpesvirusenters the cell, and the cell infected by the recombinant herpesvirusproduces proteins encoded by the viral genome, including the chimeric gBharboring the heterologous polypeptide ligand. The infected cellproduces progeny virus which lyses the cell, thereby killing it.

The term “retargeting”, as used herein, means that the recombinantherpesvirus of the present invention is targeted to the target moleculeof the ligand. However, the recombinant herpesvirus is still capable ofbeing targeted to the natural receptor of the unmodified herpesvirus.Retargeting is different from “detargeting”, which means that therecombinant herpesvirus is no longer capable of being targeted to thenatural receptor of the unmodified herpesvirus. “Detargeting” means thatthe recombinant virus is only targeted to the target molecule of theligand.

The indication of a specific amino acid number or region of gB, as usedherein, refers to the “precursor” form of gB, as exemplified in SEQ IDNO: 1 that includes the N-terminal signal sequence comprising the first30 amino acids. The “mature” form of gB starts with amino acid 31 of SEQID NO: 1 and extends until amino acid 904. As gB glycoproteins withamino acid sequences different from SEQ ID NO: 1 are also comprised bythe present invention, the indication of a specific amino acid number orof a specific amino acid region which relates to SEQ ID NO: 1 means alsothe amino acid number or region of a homologous gB, which corresponds tothe respective amino acid number or region of SEQ ID NO: 1.

The term “recombinant” herpesvirus, as referred to herein, refers to aherpesvirus that has been genetically engineered by geneticrecombination to include additional nucleic acid sequences which encodethe heterologous polypeptide. Methods of producing recombinantherpesviruses are well known in the art (see for example Sandri-Goldinet al., 2006). However, the present invention is not limited to geneticengineering methods. Also other methods may be used for producing anherpesvirus having fused or inserted a heterologous polypeptide ligandto or into gB, respectively.

The term “chimeric glycoprotein B” or “chimeric gB”, or “chimeric gB”,as used herein, means a gB having fused to or inserted into the gB aheterologous polypeptide ligand. The chimeric gB is encoded by therecombinant virus, is synthesized with the cell that produces therecombinant virus, and becomes incorporated in the envelope of thevirion. Methods to produce the recombinant viruses by geneticengineering are known in the art. Methods for producing chimericglycoprotein B are known in the art.

The term “herpesvirus”, as referred to herein, refers to a member of theHerpesviridae family of double-stranded DNA viruses, which cause latentor lytic infections. Herpesviruses all share a common structure in thattheir genomes consist of relatively large (about from 100.000 to 200.000base pairs), double-stranded, linear DNA encoding 80 to 200 genes,encased within an icosahedral protein cage called the capsid which isitself wrapped by a protein layer called the tegument containing bothviral proteins and viral mRNAs and a lipid bilayer membrane called theenvelope. This whole particle is also known as a virion. The term“herpesvirus” also refers to members of the Herpesviridae family whichare mutated comprising one or more mutated genes, such as, e.g.,herpesviruses which were modified in a laboratory.

In a preferred embodiment, the herpesvirus is selected from the groupconsisting of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2(HSV-2), Varicella Zoster Virus (human herpesvirus 3 (HHV-3)), swinealphaherpesvirus Pseudorabiesvirus (PRV), Chimpanzee alpha1 herpesvirus(ChHV), Papiine herpesvirus 2 (HVP2), Cercopithecine herpesvirus 2(CeHV2), Macacine herpesvirus 1 (MHV1), Saimiriine herpesvirus 1 (HVS1),Callitrichine herpesvirus 3 (CalHV3), Saimiriine herpesvirus 2 (HVS2),Bovine herpesvirus 1 (BoHV-1), Bovine Herpesvirus 5 (BoHV-5), Equineherpesvirus 1 (EHV-1), Equine herpesvirus 2 (EHV-2), Equine herpesvirus5 (EHV-5), Canine herpesvirus 1 (CHV), Feline herpesvirus 1 (FHV-1),Duck enteritis virus (DEV), Fruit bat alphaherpesvirus 1 (FBAHV1),Bovine herpesvirus 2 (BoHV-2), Leporid herpesvirus 4 (LHV-4), Equineherpesvirus 3 (EHV-3), Equine herpesvirus 4 (EHV-4), Equine herpesvirus8 (EHV-8), Equid herpesvirus 9 (EHV-9), Cercopithecine herpesvirus 9(CeHV-9), Suid herpesvirus 1 (SuHV-1), Marek's disease virus (MDV),Marek's disease virus serotype 2 (MDV2), Falconid herpesvirus type 1(FaHV-1), Gallid herpesvirus 3 (GaHV-3), Gallid herpesvirus 2 (GaHV-2),Lung-eye-trachea disease-associated herpesvirus (LETV), Gallidherpesvirus 1 (GaHV-1), Psittacid herpesvirus 1 (PsHV-1), Humanherpesvirus 8 (HHV-8), Human herpesvirus 4 (HHV-4), Chelonid herpesvirus5 (ChHV5), Ateline herpesvirus 3 (AtHV3) or Meleagrid herpesvirus 1(MeHV-1). In a more preferred embodiment, the herpesvirus is HSV-1 orHSV-2, most preferably HSV-1.

The term “heterologous”, as used herein, refers to a polypeptide that isnot encoded by the herpesvirus genome, or that of any other herpesvirus.Preferably, the term “heterologous” refers to a polypeptide which bindsto a cell which carries a target molecule of the ligand and is to beinfected by the recombinant herpesvirus of the present invention. Theheterologous polypeptide may be a natural polypeptide, or part thereof,or an artificial polypeptide, not found in nature.

The term “polypeptide”, as used herein, is a continuous and unbranchedpeptide chain consisting of amino acids connected by peptide bonds. Thelength of the polypeptide chain is unlimited and may range from someamino acids such as 5 amino acids to some hundreds or thousands aminoacids. In the present invention, a polypeptide may be used as a ligandor as a target molecule. The length of the chain depends on the moleculewhich is the starting molecule for the ligand or target molecule. Morethan one polypeptide chains may assemble to a complex such as anantibody. The term “polypeptide”, as used herein, also comprises anassembly of polypeptide chains. The term “peptide”, as used herein, is ashort polypeptide chain, usually consisting of less than about 50 aminoacid residues, preferably less than about 40 amino acids residues, ormore preferably of between about 10 and about 30 amino acids. Theminimum length is 5 amino acid residues.

The term “corresponding region of a homologous gB” refers to a region ofa gB which aligns with a given region of the gB according to SEQ ID NO:1 when using the Smith-Waterman algorithm and the following alignmentparameters: MATRIX: BLOSUM62, GAP OPEN: 10, GAP EXTEND: 0.5. Thisalgorithm is generally known and used in the art if performing pairwisesequence comparisons and the skilled person knows how to apply it. Incase only a part or parts of the given region of SEQ ID NO: 1 alignswith the sequence of a homologous gB using above algorithm andparameters, the term “corresponding region” refers to the region whichaligns with the part(s) of the given region of SEQ ID NO: 1. In thiscase, the region in the homologous gB, in which the ligand is inserted,comprises only the amino acids which align with the part(s) of the givenregion of SEQ ID NO: 1. The term “corresponding region” may also referto a region which is flanked by corresponding flanking sequences,wherein the flanking sequences align, using above algorithm andparameters, with sequences flanking the region of SEQ ID NO: 1. Theseflanking sequences are at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50amino acids long. Other algorithms which may be used are the algorithmsof Needleman and Wunsch, 1970, the similarity method of Pearson andLipman, 1988, or the algorithm of Karlin and Altschul, 1990, modified byKarlin and Altschul, 1993, or computerized implementations of thesealgorithms.

The term “corresponding amino acid” refers to an amino acid which ispresent within a corresponding region and which is the counterpart of agiven amino acid of SEQ ID NO: 1 in the alignment. A corresponding aminoacid must not be identical to its counterpart in SEQ ID NO: 1 in thealignment, as far as it is present within a corresponding region.

A ligand, as referred to herein, binds or is capable of binding to atarget molecule accessible on the surface of a cell. Preferably, itspecifically binds or is capable of specifically binding to a targetmolecule accessible on the surface of a cell, whereby the term“specifically binds” refers to a binding reaction wherein the ligandbinds to a particular target molecule of interest, whereas it does notbind in a substantial amount (less than 10%) to other molecules presenton cells or to other molecules to which the ligand may come in contactin an organism. Generally, a ligand that “specifically binds” a targetmolecule has an equilibrium affinity constant greater than about 10⁵(e.g., 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or more) mole/liter for thattarget molecule. Preferably, the ligand mediates the capability that thevirus fuses with the cell, so that more preferably the virus then entersthe cell, and still more preferably kills the cell. It is understoodthat the ligand is not harmful to humans. Moreover, the ligand is not aherpesvirus protein or is not derived by modification from a herpesvirusprotein.

The ligand may be a natural or artificial polypeptide ligand which iscapable of specifically binding to a target molecule which is accessibleon a cell, preferably wherein the heterologous polypeptide ligand iscapable of binding to a target molecule present on a cell present incell culture or to a target molecule present on a diseased cell. Naturalpolypeptide ligands are natural polypeptides that are capable of bindingto a target molecule. Thus, the ligand may be the natural ligand of anatural target molecule such as a receptor molecule, which is accessibleon a cell. Examples of such a ligand may be a cytokine, a chemokine, animmune checkpoint blocker, or a growth factor. Known examples are EGFand IL13. Alternatively, the ligand is an antibody that binds to atarget molecule. Still alternatively, the ligand is a naturalpolypeptide which has been selected to bind to an artificial targetmolecule, whereby the target molecule is designed to be capable ofbinding to the ligand. The natural polypeptide may be derived from anyorganism, preferably from an organism which is not harmful to human. Forexample, the natural polypeptide is a fungal or bacterial polypeptide,such as a polypeptide from the genus Saccharomyces such as Saccharomycescerevisiae. An example of a natural polypeptide is the GCN4 yeasttranscription factor. Artificial polypeptide ligands have non-naturallyoccurring amino acid sequences that function to bind a particular targetmolecule. The sequence of the artificial polypeptide ligand may bederived from a natural polypeptide which is modified, includinginsertion, deletion, replacement and/or addition of amino acids, wherebythe binding capability of the corresponding natural polypeptide isretained. For example, the ligand may be a part of a naturalpolypeptide, as referred to above, as far as said part is capable ofbinding to the target molecule to which the corresponding full-lengthpolypeptide binds. Alternatively, the natural polypeptide has beenmodified to comprise an amino acid identity to the corresponding naturalpolypeptide of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%,whereby the modified polypeptide retains the activity of thecorresponding natural polypeptide, such as binding to the targetmolecule. Still alternatively, the artificial polypeptide ligand mayhave 274 amino acid residues or less, preferably less than 200 aminoacid residues, more preferably less than 50 amino acid residues, stillmore preferably less than 40 amino acids residues, or still morepreferably between 10 and 30 amino acids, most preferably 20 aminoacids, such as a part of a natural polypeptide or a peptide from a(random) peptide library. Still alternatively, the polypeptide is anantibody derivative or an antibody mimetic that binds to the targetmolecule. The antibody, antibody derivative or antibody mimetic may bemono-specific (i.e. specific to one target molecule accessible on thesurface of a cell) or multi-specific (i.e. specific to more than onetarget molecule accessible on the surface of the same or a differentcell), for example bi-specific or tri-specific (e.g., Castoldi et al.,2013, Castoldi et al., 2012). Specificity of the virus is increased bysimultaneously targeting more than one target molecule on the same cell.If more than one target molecule present on different cells aretargeted, tumor heterogeneity can be addressed.

The term “antibody derivative”, as referred to herein, refers to amolecule comprising at least one antibody variable domain, but notcomprising the overall structure of an antibody. The antibody derivativeis still capable of binding a target molecule. Preferably, the antibodyderivative mediates the capability that the virus fuses with the cell,so that more preferably the virus then enters the cell, and still morepreferably kills the cell. Said derivatives may be antibody fragmentssuch as

Fab, Fab2, scFv, Fv, or parts thereof, or other derivatives orcombinations of immunoglobulins such as nanobodies, diabodies,minibodies, camelid single domain antibodies, single domains or Fabfragments, domains of the heavy and light chains of the variable region(such as Fd, VL, including Vlambda and Vkappa, VH, VHH) as well asmini-domains consisting of two beta-strands of an immunoglobulin domainconnected by at least two structural loops. Preferably, the antibodyderivative is a single chain antibody, more preferably scFv which is afusion protein of the variable regions of the heavy (V_(H)) and lightchains (V_(L)) of immunoglobulins, connected with a short linkerpeptide. The N-terminus of V_(H) is either connected with the C-terminusof V_(L) or the N-terminus of V_(L) is connected with the C-terminus ofV_(H).

The term “antibody mimetic”, as referred to herein, refers to organiccompounds that, like antibodies, can specifically bind antigens, butthat are not structurally related to antibodies. They are usuallyartificial peptides or proteins with a molar mass of about 3 to 20 kDa.They may have therapeutic or diagnostic effects. Non-limiting examplesof antibody mimetics are affibodies, affilins, affimers, affitins,anticalins, avimers, DARPins, fynomers, Kunitz domain peptides,monobodies, Z domain of Protein A, Gamma B crystalline, ubiquitin,cystatin, Sac7D from Sulfolobus acidocaldarius, lipocalin, A domain of amembrane receptor, ankyrin repeat motive, SH3 domain of Fyn, Kunitsdomain of protease inhibitors, the 10^(th) type III domain offibronectin, synthetic heterobivalent or heteromultivalent ligands(Josan et al., 2011, Xu et al., 2012, Shallal et al., 2014).

The term “a heterologous polypeptide ligand” or “a ligand”, as referredto herein, includes one or more than one ligand(s) such as 2, 3, or 4ligands. This means that the recombinant herpesvirus may comprise oneligand or may comprise more than one ligand. The presence of one ligandallows the targeting of one target cell type. If more than one ligand ispresent, the ligands may be fused to or inserted into one gB beinglocated in the gB molecule on different sites or on the same site, i.e.successively, or the ligands may be fused to or inserted into differentgBs. Alternatively, if more than one ligand are present, the second orfurther ligand(s) may be comprised by a glycoprotein of the herpesvirusother than gB, such as gD and/or gH. The different ligands may targetdifferent target molecules present on the same target cell or ondifferent target cells, preferably on different target cells. In analogyto the above, the term “a target molecule”, as referred to herein,includes one or more than one target molecule(s) such as 2, 3, or 4target molecules. Consequently, the recombinant herpesvirus may bind toone target cell or may bind to more than one target cells such as 2, 3,or 4 different cells.

In a preferred embodiment of the present invention, the heterologouspolypeptide ligand is an artificial polypeptide, preferably an scFv,which is capable of binding to a natural receptor on a diseased cell,preferably a tumor cell, more preferably a tumor cell expressing HER2,such as a breast cancer cell, ovary cancer cell, stomach cancer cell,lung cancer cell, head and neck cancer cell, osteosarcoma cell,glioblastoma multiforme cell, or salivary gland tumor cell. In a morepreferred embodiment, the heterologous polypeptide ligand is scFvcapable of binding to HER2. In the most preferred embodiment, theheterologous polypeptide ligand is scFv as identified by SEQ ID NO: 32.

In an additionally or alternatively preferred embodiment of the presentinvention, the heterologous polypeptide ligand is an artificialpolypeptide, preferably a part of a natural polypeptide, which iscapable of binding to an artificial target molecule present on a cellpresent in cell culture. The length of the ligand is more preferablyless than about 50 amino acid residues, still more preferably less thanabout 40 amino acids residues, still more preferably of between about 10and about 30 amino acids, or most preferably 20 amino acids. The ligandand target molecules are specifically constructed to bind to each other.More preferred, the heterologous polypeptide ligand is a part of theGCN4 yeast transcription factor. Still more preferred, the heterologouspolypeptide ligand is the part of the GCN4 yeast transcription factor ascomprised by SEQ ID NO: 37, most preferably, the ligand is the moleculeidentified by the sequence of SEQ ID NO: 37. In an alternativeembodiment, the ligand may be the molecule identified by the sequence ofSEQ ID NO: 38.

In a more preferred embodiment of the present invention, the preferredand alternatively preferred embodiment, as mentioned above, arecombined. Namely, the recombinant herpesvirus of the present inventionsimultaneously comprises two heterologous polypeptide ligands, one beingcapable of binding to a diseased cell and one being capable of bindingto a cell present in cell culture.

The GCN4 yeast transcription factor used as a polypeptide ligand fusedto or inserted into gB is state of the art (see e.g. Arndt and Fin,1986; Hope and Struhl, 1987). An exemplary GCN4 yeast transcriptionfactor is one identified by SEQ ID NO: 43 (UniProtKB—P03069 (GCN_YEAST),as encoded by AJ585687.1 (SEQ ID NO: 42). The term “GCN4 yeasttranscription factor”, as referred to herein, refers to any GCN4 yeasttranscription factor present in nature. Alternatively, GCN4 yeasttranscription factor, as referred to herein, refers to any GCN4 yeasttranscription factor which has an amino acid identity to the sequence ofSEQ ID NO: 43 of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100%. Alternatively, the GCN4 yeast transcription factor, as referred toherein, refers to any GCN4 yeast transcription factor which has an aminoacid homology to SEQ ID NO: 43 of at least 50%, 60%, 70%, 80%, 85%, 90%,95%, or 100%. A GCN4 yeast transcription factor is “homologous” or a“homolog” if it has an identity to SEQ ID NO: 43 of at least 70%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, if it has an amino acidhomology to SEQ ID NO: 43 of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,or 100%, or if it has the same activity as the GCN4 yeast transcriptionfactor according to SEQ ID NO: 43. Preferably, “same activity” may beunderstood in the sense that GCN4 yeast transcription factor works as atranscription factor in the same way as the GCN4 yeast transcriptionfactor according to SEQ ID NO: 43. The term “part thereof” comprises anypart of the GCN4 yeast transcription factor against which a targetmolecule can be generated to which the “part thereof” is capable ofbinding. Preferably, the length of “the part thereof” is thus that aligand length of 274 amino acids or less, preferably less than 200 aminoacid residues, more preferably less than 50 amino acids residues, stillmore preferably less than 40 amino acids residues, still more preferablybetween 10 and 30 amino acids, or still more preferably 20 amino acidsresults, whereby the ligand may include additional sequences such aslinker sequences.

The most preferred “part thereof” is the sequence YHLENEVARLKK (SEQ IDNO: 38) of GCN4 yeast transcription factor to which two flanking wt(wildtype) GCN4 residues may be added on each side. For fusion to orinsertion into gB, a GS linker may be additionally present on each sideof the peptide. This construct is herein named GCN4 peptide (SEQ ID NO:37). This 20 amino acid peptide confers to the herpesvirus the abilityto infect and replicate in a cell line bearing a target molecule towhich the “part thereof” binds.

In the recombinant herpesvirus of the present invention, the ligand maybe fused to or inserted into gB, between amino acids 43 and 44 of gB,corresponding to amino acids 13 and 14 of mature gB (SEQ ID NO: 2). Inthis context, the term “fused” or “fusion”, as referred to herein,refers to the addition of the polypeptide ligand to the N-terminal aminoacid of gB by peptide bonds, either directly or indirectly via a peptidelinker. “Fused” or “fusion” to the N-terminal region is different from“insertion” insofar as “fused” or “fusion” means addition to theterminus of gB, whereas “insertion” means incorporation into the gB.

A peptide linker, as referred to herein, serves to connect, within apolypeptide, polypeptide sequences derived from different sources. Sucha linker serves to connect and to enable proper folding of theheterologous polypeptide ligand with glycoprotein B sequences or toconnect ligand portions within the heterologous polypeptide ligand. Itmay also serve to connect ligand sequences with glycoprotein sequencesother than gB. A linker has typically a length between 1 and 30 aminoacids, preferably 5 to 25 amino acids, more preferably 8 to 20 aminoacids, such as 8, 12 or 20 amino acids and may comprise any amino acids.Preferably, it comprises the amino acid(s) Gly and/or Ser and/or Thr,more preferably it comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 amino acids selected from the group consisting of Gly, Ser and/orThr. Most preferably, it consists of the amino acids Gly and/or Ser.Linkers based on Gly and/or Ser provide flexibility, good solubility andresistance to proteolysis. Alternatively, the linker may notpredominantly comprise glycine, serine and/or threonine, but glycine,serine and/or threonine may not be present or only to a minor extent.

In the recombinant herpesvirus of the present invention, the ligand maybe alternatively inserted at any amino acid within a disordered regionof gB, whereby the ligand is not inserted at any amino acid within theregion spanning from amino acids 77 to 88 of gB according to SEQ ID NO:1 or a corresponding region of a homologous gB. However, a ligand ofshort length not exceeding 120 amino acids may be inserted within theregion spanning from amino acids 77 to 88 of gB according to SEQ ID NO:1 or a corresponding region of a homologous gB. As referred to herein,disordered region is meant to comprise a region which lacks a fixedtertiary structure, is unstructured, and consequently not amenable tobeing resolved in a crystal structure. Often, disordered regions areextended, i.e. random coil like, or collapsed, i.e. molten globule like.Disordered structures in gB are referred to in Gallagher et al., 2014,Heldwein et al., 2006, and Lin et al., 2007 and may be present in theN-terminal region (extending from amino acids 31 to 108), in a centralregion (extending from amino acids 460 to 491) and the C-terminal region(extending from amino acids 796 to 904) (Heldwein et al., 2006) of HSVgB. Positions which are mentioned as being disordered are amino acids 31to 108 (N-terminal region) (Lin et al., 2007), amino acids 460 to 491(central region) (Heldwein et al., 2006) and amino acids 796 to 904(C-terminal region) (Heldwein et al., 2006, Lin et al. 2007) of HSV-1gB. Preferably, the ligand is inserted at any amino acids within aregion spanning from amino acids 31 to 108 or 460 to 491 of gB accordingto SEQ ID NO: 1 or a corresponding region of a homologous gB. The ligandis not inserted within the region spanning from amino acids 77 to 88 ofgB according to SEQ ID NO: 1 or a corresponding region of a homologousgB. However, a ligand of short length not exceeding 120 amino acids maybe inserted within the region spanning from amino acids 77 to 88 of gBaccording to SEQ ID NO: 1 or a corresponding region of a homologous gB.

The ligand may be alternatively inserted at any amino acid within aregion spanning from amino acids 31 to 77 or 88 to 184, preferably aminoacids 31 to 77 or 88 to 136 or more preferably 31 to 77 or 88 to 108, orinto a region spanning from amino acids 409 to 545, preferably aminoacids 459 to 545, more preferably amino acids 459 to 497, or still morepreferably amino acid 460 to 491, of gB according to SEQ ID NO: 1 orwithin a corresponding region of a homologous gB. Moreover, a ligand ofshort length not exceeding 120 amino acids may be inserted within theregion spanning from amino acids 77 to 88 of gB according to SEQ ID NO:1 or a corresponding region of a homologous gB. These regions includeamino acids which are located within disordered regions of gB, however,also include amino acids which are located in the neighborhood ofdisordered regions. The regions, as indicated above, have been found toaccept polypeptide insertions, thereby maintaining the capability of gBto function as a fusogen mediating membrane fusions of cells carrying gBand the receptor (Gallagher et al., 2014; Lin and Spear, 2007; Potel etal., 2002).

The term “inserted” or “insertion”, as referred to herein in the sensethat a ligand is inserted into gB, refers to the incorporation of thepolypeptide ligand into the gB, wherein the incorporated polypeptide isintroduced between two amino acids of the gB by peptide bonds, eitherdirectly or indirectly via one or more peptide linkers, morespecifically via an upstream and/or downstream located peptide linkerwith respect to the insert. The linker is directly connected to thepolypeptide ligand. The fusion of a polypeptide ligand to gB can also beseen as an insertion of the polypeptide ligand sequence into the gBprecursor, exemplified by SEQ ID NO: 1 or a homologous gB, directlybefore amino acid 1 of the gB; such an insertion is herein termed asfusion. The gB carrying the fused, or inserted polypeptide is hereinreferred to chimeric gB. The chimeric gB is part of the virion envelope.The definition of “linker” is, as described above.

The insertion and fusion are preferably carried out by geneticengineering of the gB gene, in the genome of HSV. The geneticengineering of HSV genomes is known in the art, exemplified by, but notlimited to, BAC technologies

The present inventors have found that insertion of a heterologouspolypeptide ligand at an amino acid within the region of amino acids 77to 88 of the gB, as exemplified by SEQ ID NO: 3, in which the scFv toHER2 is inserted between amino acids 81 to 82, does not result in theretargeting of the recombinant herpesvirus to cells carrying thereceptor of the ligand. The present inventors believe that the reasonfor this lack of retargeting is the presence of a proline-rich region(PPPPXP) and a predicted N-glycosylation site (NAT) within this region.As proline disrupts protein secondary structure and/or imposes its ownkind of secondary structure with a confined phi angle that overridesother forms of secondary structure (Morgan and Rubistein, 2013)), and aspolyproline helices can induce sharp turns in the local geometry, thepolyproline stretch in this region may have constrained the conformationadopted by the ligand. In addition, the N-glycosylation site in thisregion may have shielded the ligand. Consequently, the ligand may not besufficiently available for interaction with its receptor on a cellsurface. Moreover, the present inventors have also found that insertionof a heterologous polypeptide ligand at any amino acid within the regionspanning from amino acids 77 to 88 of the gB results in the retargetingof the recombinant herpesvirus to cells carrying the receptor of theligand, if the heterologous polypeptide ligand is of short length. Thus,the present invention provides a recombinant herpesvirus comprising aheterologous polypeptide ligand capable of binding to a target moleculeand inserted into glycoprotein B (gB) present in the envelope of theherpesvirus, wherein the ligand is of short length and is inserted atany amino acid within a region spanning from amino acids 77 to 88 of gBaccording to SEQ ID NO: 1 or within a corresponding region of ahomologous gB. “Short length” means a length which does not exceed 120amino acids, such as 5 to 120 amino acids, 5 to 110, 5 to 100, 5 to 90,5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 10 to 30,10 to 20, 20 or 12 amino acids. Preferably, the ligand has a length of10 to 30 amino acids, more preferably the ligand has a length of 12 to20 amino acids, still more preferably the ligand is 12 or 20 aminoacids. Insertion may be at any amino acid between amino acids 77 to 88,such as behind amino acid 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, or 87.Preferably, the ligand is inserted between amino acids 81 and 82. Any ofa combination of a ligand of a length as indicated above inserted behindany of the amino acids mentioned above results in the retargeting of theherpesvirus to the target molecule of the ligand. Preferably, theheterologous polypeptide ligand may be a part of a natural polypeptidecapable of binding to a target molecule present on a cell present incell culture, such as a part of the GCN4 yeast transcription factor,such as the part of the GCN4 yeast transcription factor as comprised bySEQ ID NO: 37. Most preferably, the ligand is the molecule identified bythe sequence of SEQ ID NO: 37. Such a ligand may be inserted at anyamino acid within the region spanning from amino acids 77 to 88,preferably between amino acids 81 to 82. Most preferably, the ligand isthe molecule identified by the sequence of SEQ ID NO: 37 insertedbetween amino acids 81 and 82 of gB. The amino acid numbers refer to SEQID NO: 1 or corresponding amino acids of a homologus gB.

As used herein, the target molecule may be any molecule which isaccessible on the surface of a cell and which can be bound by theheterologous polypeptide ligand. The target molecule may be a naturalmolecule such as a polypeptide or protein, a glycolipid or a glycoside.For example, the target molecule may be a receptor, such as a proteinreceptor. A receptor is a molecule embedded in a membrane of a cell thatreceives chemical signals from the outside via binding of a ligand,causing some form of a cellular response. Alternatively, the targetmolecule may be a molecule that is a drug target, such as enzymes,transporters or ion-channels, present on the surface of a cell.Preferably, the target molecule is present on a diseased cell or on acell present in cell culture. Preferred target molecules are those whichare naturally present on diseased cells of an organism, such asmentioned below, in a specific or abnormal manner. “Specific manner” maybe understood in the sense that the target molecule is overexpressed onthe diseased cell, whereas it is not or only to a minor extent, i.e. toan extent to which it is usually present on a respective normal cell,expressed on the normal cell. “Abnormal manner” may be understood in thesense that the target molecule is present on a diseased cell in amutated form, as compared to the respective molecule of the respectivenon-diseased cell. Therefore, retargeting a herpesvirus to a targetmolecule, such as a specifically expressed or mutated target molecule,results in a higher infection and eradication rate of a cell carryingthe target molecule as compared to a cell that does not carry the targetmolecule or carries the target molecule at a lower level or carries thewildtype (non-mutated) target molecule.

Alternatively, the target molecule may be an artificial molecule. Theterm “artificial target molecule”, as referred to herein, is a moleculethat does not naturally occur, i. e. that has a non-natural amino acidsequence. Such artificial molecule may be constructed to be expressed bya cell on its surface, as e.g. described in Douglas et al., 1999; andNakamura et al., 2005 or it may be bound by a cell surface. Artificialtarget molecules have non-naturally occurring amino acid sequences thatfunction to bind a particular ligand. Examples of artificial targetmolecules are antibody derivatives, or antibody mimetics. Artificialtarget molecules are preferably present on the surface of a cell presentin cell culture which may be used for producing the recombinantherpesvirus. Preferred artificial target molecules present on a cellpresent in cell culture are scFvs. In the context of artificial targetmolecule, antibodies are comprised by the term “artificial targetmolecule” which may be present on a cell present in cell culture bywhich they are not naturally produced.

In a preferred embodiment, the target molecule is a tumor-associatedreceptor, preferably a member of the EGF receptor family, includingHER2, EGFR, EGFRIII or EGFR3 (ERBB3), EGFRvIII, MET, FAP, PSMA, CXCR4,CEA, CADC, Mucins, Folate-binding protein, GD2, VEGF receptors 1 and 2,CD20, CD30, CD33, CD52, CD55, the integrin family, IGF1R, the Ephrinreceptor family, the protein-tyrosine kinase (TK) family, RANKL,TRAILR1, TRAILR2, IL13Ralpha, UPAR, Tenascin, PD-1, PD-L1, CTL-A4, andadditional members of the immune checkpoint family regulators,tumor-associated glycoprotein 72, ganglioside GM2, A33, Lewis Y antigen,or MUC1, most preferably HER2. Preferably, the target molecule is HER2which is overexpressed by some tumor cells such as breast cancer cells,ovary cancer cells, stomach cancer cells, lung cancer cells, head andneck cancer cells, osteosarcoma cells, glioblastoma multiforme cells, orsalivary gland tumor cells, but is expressed at very low levels innon-malignant tissues. A tumor-associated receptor is a receptor whichis expressed by a tumor cell in a specific or abnormal manner.Alternatively, the target molecule is a molecule derived from aninfectious agent such as a pathogen (e.g. a virus, bacterium orparasite) that has infected a cell. The target molecule is expressed onthe surface of the infected cell (such as HBsAg from HBV, gpI20 fromHIV, E1 or E2 from HCV, LMP1 or LMP2 from EBV). The pathogen may resultin an infectious disease, such as a chronic infectious disease. Stillalternatively, the target molecule is expressed by a degenerativedisorder-associated cell or by a senescent cell such as CXCR2 or theIL-1 receptor. In another preferred embodiment, the target molecule isan antibody derivative, more preferably an scFv, still more preferablyan scFv capable of binding to a part of the GCN4 yeast transcriptionfactor, still more preferably an scFv capable of binding to the part ofthe GCN4 yeast transcription factor as comprised by SEQ ID NO: 37, stillmore preferably the scFv as comprised by SEQ ID NO: 39, most preferablythe molecule identified by the sequence of SEQ ID NO: 41.

The term “cell”, as referred to herein, is any cell which carries atarget molecule and which can be infected by the recombinant herpesvirusof the present invention. The cell may be a naturally occurring cellwhich is unwanted and shall be eliminated, such as a diseased cell.Examples of diseased cells are given below. Preferred diseased cells arethose comprising HER2. Alternatively, the cell may be a cell whichserves to produce the recombinant herpesvirus. Such cell may be any cellwhich can be infected by the recombinant herpesvirus of the presentinvention and which can produce the herpesvirus. Moreover, propagationof the herpesvirus shall be avoided in diseased cells, so as to avoidthe introduction of material such as DNA, RNA and/or protein of diseasedcells such as tumor cells in humans, Therefore, the cell for producingthe herpesvirus is a cell which is not harmful if present in humans,e.g. a non-diseased cell. The cell may be present as a cell line. Forproducing the recombinant herpesvirus, the cell is present in cellculture. Therefore, a cell which serves to produce the recombinantherpesvirus is termed herein “cell present in cell culture”. Thus, thecell may be a cultured cell suitable for growth of herpesvirus,preferably the cell is a cell line approved for herpesvirus growth.Examples of such cells are Vero, 293, 293T, HEp-2, HeLa, BHK, or RScells, preferably Vero cells. Preferably, the cell present in cellculture has been modified to express a target molecule which is notnaturally expressed by the corresponding parent cell or the cell presentin cell culture has been modified and binds the target molecule on itssurface. More preferably, the cell comprises as the target molecule anantibody derivative, still more preferably an scFv, still morepreferably an scFv capable of binding to a part of the GCN4 yeasttranscription factor, still more preferably an scFv capable of bindingto the part of the GCN4 yeast transcription factor as comprised by SEQID NO: 37, still more preferably the scFv as comprised by SEQ ID NO: 39,most preferably the molecule identified by the sequence of SEQ ID NO:41.

A “cultured” cell is a cell which is present in an in vitro cell culturewhich is maintained and propagated, as known in the art. Cultured cellsare grown under controlled conditions, generally outside of theirnatural environment. Usually, cultured cells are derived frommulticellular eukaryotes, especially animal cells. “A cell line approvedfor growth of herpesvirus” is meant to include any cell line which hasbeen already shown that it can be infected by a herpesvirus, i. e. thevirus enters the cell and is able to propagate and produce the virus. Acell line is a population of cells descended from a single cell andcontaining the same genetic composition. Preferred cells for propagationand production of the recombinant herpesvirus are Vero, 293, 293T,HEp-2, HeLa, BHK, or RS cells.

The term “diseased cell”, as used herein, refers to a cell whichnegatively influences an organism and is, therefore, not wanted. Theeradication of such a cell is desired, as its killing may be live-savingor enhances the health of an organism. In a preferred embodiment, thediseased cell is characterized by an abnormal growth, more preferablythe cell is a tumor cell. In an alternative preferred embodiment, thecell is an infected cell such as a chronically infected cell, adegenerative disorder-associated cell or a senescent cell.

In case of a tumor cell, the underlying disease is a tumor, preferablyselected from the group consisting of adrenal cancer, anal cancer, bileduct cancer, bladder cancer, bone cancer, brain/CNS tumors, breastcancer, cancer of unknown primary treatment, Castleman disease, cervicalcancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewingfamily of tumors, eye cancer, gallbladder cancer, gastrointestinalcarcinoid tumors, gastrointestinal stromal tumor (gist), gestationaltrophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer,laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lungcancer, lymphoma, lymphoma of the skin, malignant mesothelioma, multiplemyeloma, myelodysplastic syndrome, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oralcavity and oropharyngeal cancer, osteosarcoma, ovarian cancer,pancreatic cancer, penile cancer, pituitary tumors, prostate cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma—adultsoft tissue cancer, skin cancer, small intestine cancer, stomach cancer,testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma,vaginal cancer, vulvar cancer, Waldnstrom macroglobulinemia, and Wilmstumor. Preferred tumor diseases are HER2-positive cancers (like breastcancer, ovary cancer, stomach cancer, lung cancer, head and neck cancer,osteosarcoma and glioblastoma multiforme), EGFR-positive cancers (likehead and neck cancer, glioblastoma multiforme, non-small cell lungcancer, breast cancer, colorectal and pancreatic cancer),EGFR-vIII-positive cancers (like glioblastoma multiforme), PSMA-positivecancers (like prostate cancer), CD20+ positive lymphoma, and EBV relatedtumors such as B-cell lymphoproliferative disorders such as Burkitt'slymphoma, classic Hodgkin's lymphoma, and lymphomas arising inimmunocompromised individuals (post-transplant and HIV-associatedlymphoproliferative disorders), T-cell lymphoproliferative disorders,angioimmunoblastic T-cell lymphoma, extranodal nasal type naturalkiller/T-cell lymphoma.

In case of an infected cell, the underlying disease is an infectiousdisease, such as a chronic infectious disease, wherein the infectiousagent may be a virus, a bacterium or a parasite. Examples aretuberculosis, malaria, chronic viral hepatitis (HBV, Hepatitis D virusand HCV), acquired immune deficiency syndrome (AIDS, caused by HIV,human immunodeficiency virus), EBV related disorders, or HCMV relateddisorders.

In case of a degenerative disorder-associated cell, the underlyingdisease may be Alzheimer's disease, amyotrophic lateral sclerosis (ALS),Lou Gehrig's Disease, osteoarthritis, atherosclerosis, Charcot MarieTooth disease (CMT), chronic obstructive pulmonary disease (COPD),chronic traumatic encephalopathy, diabetes, ehlers-danlos syndrome,essential tremor, Friedreich's ataxia, huntington's disease,inflammatory bowel disease (IBD), keratoconus, keratoglobus, maculardegeneration, marfan's syndrome, multiple sclerosis, multiple systematrophy, muscular dystrophy, Niemann Pick disease, osteoporosis,Parkinson's Disease, progressive supranuclear palsy, prostatitis,retinitis pigmentosa, rheumatoid arthritis, or Tay-Sachs disease. Theterm “degenerative disorder-associated cell” refers to a cell which isin relationship with the disorder, meaning that an alteration of thecell contributes to the development of the disease or the cell isaltered as a consequence of the disease. Destroying the cell results inthe treatment of the disease.

In case of a senescent cell, the underlying disease is asenescence-associated disease, such as (i) rare genetic diseases calledprogeroid syndromes, characterized by pre-mature aging: Werner syndrome(WS), Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), Cockaynesyndrome (CS), xeroderma pigmentosum (XP), trichothiodystrophy orHutchinson-Gilford Progeria syndrome (HGPS) or (ii) common age relateddisorders, such as obesity, type 2 diabetes, sarcopenia, osteoarthritis,idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease,cataracts, neurodegenerative diseases, systemic autoimmune diseases(systemic lupus erythematosus, rheumatoid arthritis, or Sjögrensyndrome), or multiple sclerosis.

The recombinant herpesvirus of the present invention may, in addition tothe chimeric gB, comprise a modified gD glycoprotein, as disclosed in WO2009/144755, but not limited to those types of modifications. A modifiedgD may carry a deletion of the amino acid portion 6 to 38 of mature gD(as exemplified by SEQ ID NO: 5; an exemplary gD wildtype precursorsequence is indicated in SEQ ID NO: 4). Alternatively, a modified gD maycarry other modifications that detarget herpesvirus tropism from thenatural receptors Nectin-1 and HVEM. gD may, alternatively or inaddition to the modifications that detarget herpesvirus tropism, encodeadditional sequences that readdress the tropism of the herpesvirus toselected receptors of choice, which are receptors on diseased cells suchas the HER2 receptor, as described in recombinant R-LM113 (SEQ ID NO:6). Modification of gD occurs by fusing to or inserting into gD theheterologous polypeptide ligands, as defined herein. The recombinantherpesvirus of the present invention may, in addition to the chimericgB, comprise a modified gH glycoprotein, capable to retarget gH to atarget receptor molecule, comprising the heterologous polypeptideligands, as defined herein. The recombinant herpesvirus of the presentinvention may, in addition to the chimeric gB, comprise a modified gDand a modified gH glycoprotein, as disclosed in Gatta et al., 2015, butnot limited to those descriptions. Both documents are hereinincorporated by reference. The modification(s) of gD and/or gH serve(s)for readdressing the tropism of the herpesvirus to diseased cells, asdefined herein.

Thus, in an embodiment of the present invention, the recombinantherpesvirus of the present invention comprises a chimeric gB comprisinga ligand which binds to a target molecule accessible on a cell such as adiseased cell, e.g. a cell expressing HER2, or a cell present in cellculture, and a modified gD, whereby the modification may be a deletionof amino acids 6 to 38. The gD may alternatively or in addition bemodified by the insertion of a heterologous polypeptide ligand, asdefined herein, capable of retargeting the herpesvirus to a diseasedcell. Additionally or alternatively, the recombinant herpesvirus maycomprise a modified gH which comprises a heterologous polypeptideligand, as defined herein, capable of retargeting the herpesvirus to adiseased cell.

Moreover, the present inventors have found that deletion of amino acids30 to 38 or a subset thereof from gD with regard to mature gD accordingto SEQ ID NO: 62 or a corresponding region of a homologous gD results ina recombinant herpesvirus that is detargeted from the natural receptorof unmodified gD. Thus, in an embodiment of the present invention, therecombinant herpesvirus comprises a heterologous polypeptide ligand asdefined herein fused to or inserted into gB as defined herein andretargeted to the target molecule of the ligand and a gD, wherefromamino acids 30 to 38 or a subset thereof of a gD with regard to maturegD according to SEQ ID NO: 62 or a corresponding region of a homologousgD, preferably amino acids 30 and/or 38, more preferably amino acids 30and 38 are deleted, with regard to mature gD according to SEQ ID NO: 62or a corresponding region of a homologous gD.

Instead of deleted amino acids 30 to 38 or a subset thereof aheterologous polypeptide ligand, as defined herein, may be inserted,resulting in the detargeting of the recombinant herpesvirus from thenatural receptor of unmodified gD and retargeting to the target moleculeof the ligand. In addition to the replacement of a subset by aheterologous polypeptide ligand, an additional amino acid or range ofamino acids within amino acids 30 to 38 may be deleted. Thus, in apreferred embodiment amino acids 30 and 38 are deleted and aheterologous polypeptide ligand is inserted instead of amino acid 30 or38. More preferably, amino acids 30 and 38 are deleted and aheterologous polypeptide ligand is inserted instead of amino acid 38.

The term “subset thereof” means one amino acid or at least 2, such as 2,3, 4, 5, 6, 7, or 8 adjacent amino acids out of the region consisting ofamino acids 30 to 38. Thus, “subset thereof” may mean amino acids 30,31, 32, 33, 34, 35, 36, 37, or 38, 30 to 31, 30 to 32, 30 to 33, 30 to34, 30 to 35, 30 to 36, 30 to 37, 30 to 38, 31 to 32, 31 to 33, 31 to34, 31 to 35, 31 to 36, 31 to 37, 31 to 38, 32 to 33, 32 to 34, 32 to35, 32 to 36, 32 to 37, 32 to 38, 33 to 34, 33 to 35, 33 to 36, 33 to37, 33 to 38, 34 to 35, 34 to 36, 34 to 37, 34 to 38, 35 to 36, 35 to37, 35 to 38, 36 to 37, 36 to 38, 37 to 38. The term “subset” maycomprise one or more subsets, such as 2, 3, 4, or 5 subsets. Forexample, “subset” may comprise amino acid 30 and amino acid 38, thedeletion thereof resulting in a gD that does not comprise amino acids 30and 38. Deletion of a subset, or the whole of amino acids 30 to 38,results in the inactivation of the nectin-1 binding site of gD reducingthe binding capability of gD to nectin-1 and/or in the inactivation ofthe HVEM binding site of gD reducing the binding capability of gD toHVEM. For example, if amino acid 30 is deleted, the HVEM binding site ofgD is inactivated, while the deletion of amino acid 38 results in theinactivation of the nectin-1 binding site. Deletion of both amino acids30 and 38 results in the inactivation of the HVEM binding site and thenectin-1 binding site.

The heterologous polypeptide ligand, inserted instead of amino acids 30to 38 or a subset thereof, may be a ligand capable of binding to atarget molecule present on a diseased cell, preferably an scFv capableof binding to a target molecule present on a cancer cell, such as abreast cancer cell, ovary cancer cell, stomach cancer cell, lung cancercell, head and neck cancer cell, osteosarcoma cell, glioblastomamultiforme cell, or salivary gland tumor cell, such as HER2, morepreferably an scFv identified by SEQ ID NO: 32, whereby the targetmolecule is HER2 present on a tumor cell expressing the HER2.

In a particularly preferred embodiment of the present invention, aheterologous polypeptide ligand capable of binding to a target moleculepresent on a cell present in cell culture is inserted into gB betweenamino acids 43 to 44 and a heterologous polypeptide ligand capable ofbinding to a target molecule present on a diseased cell is insertedinstead of amino acid 38 of gD from which furthermore amino acid 30 isdeleted. Most preferably, the heterologous polypeptide ligand capable ofbinding to a target molecule present on a cell present in cell culturecomprises the sequence identified by SEQ ID NO: 37, the target moleculeis the molecule with the sequence identified by SEQ ID NO: 41, and thecell present in cell culture expresses the molecule identified by thesequence of SEQ ID NO: 41, and the heterologous polypeptide ligandcapable of binding to a target molecule present on a diseased cellcomprises an scFv identified by SEQ ID NO: 32, the target molecule isHER2, and the cell is a tumor cell expressing HER2, preferably a breastcancer cell, ovary cancer cell, stomach cancer cell, lung cancer cell,head and neck cancer cell, osteosarcoma cell, glioblastoma multiformecell, or salivary gland tumor cell.

Moreover, disclosed is a recombinant herpesvirus comprising a gD,wherefrom amino acids 30 to 38 or a subset thereof with regard to maturegD according to SEQ ID NO: 62 or a corresponding region or correspondingamino acids of a homologous gD, preferably amino acids 30 and/or 38,more preferably amino acids 30 and 38 are deleted. Instead of deletedamino acids 30 to 38 or a subset thereof a heterologous polypeptideligand, as defined herein, may be inserted, resulting in the detargetingof the recombinant herpesvirus from the natural receptor of unmodifiedgD and retargeting to the target molecule of the ligand. In addition tothe replacement of a deleted amino acid or range of deleted amino acidsby a heterologous polypeptide ligand, an additional amino acid or rangeof amino acids within amino acids 30 to 38 may be deleted. Thus, forexample amino acids 30 and 38 are deleted and a heterologous polypeptideligand is inserted instead of amino acid 30 or 38. For example, aminoacids 30 and 38 are deleted and a heterologous polypeptide ligand isinserted instead of amino acid 38.

The amino acid numbers with respect to gD refer to mature gD accordingto SEQ ID NO: 62 or corresponding amino acids of a homologous gD. Thus,amino acid 30 with regard to mature gD according to SEQ ID NO: 62corresponds to amino acid 55 and amino acid 38 with regard to mature gDaccording to SEQ ID NO: 62 corresponds to amino acid 63 according to SEQID NO: 4 (precursor form). The amino acid numbers with respect to gBrefer to gB according to SEQ ID NO: 1 (precursor form) or correspondingamino acids of a homologous gB.

gD homologs are found in some members of the alpha subfamily ofHerpesviridae. Therefore, the term “homologous gD”, as referred toherein, refers to any gD homolog found in the gD-encoding members ofHerpesviridae. Alternatively, homologous gD, as referred to herein,refers to any gD, precursor or mature, which has an amino acid identityto the sequence of SEQ ID NO: 4 or 62, respectively, of at least 70%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. Alternatively,homologous gD, as referred to herein, refers to any gD, precursor ormature, which has an amino acid homology to SEQ ID NO: 4 or 62,respectively, of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%.The homologous gD, as referred to herein, also includes a fragment ofgD. Preferably, homologous gD, as referred to herein, including any gDfound in Herpesviridae, any gD, precursor or mature, having an aminoacid identity or homology, as defined above, to the sequence of SEQ IDNO: 4 or 62, respectively, and any fragment of a gD, has the sameactivity of the gD according to SEQ ID NO: 4 or 62. More preferably,during the entry process of the virus into a cell, gD binds to one ofits receptors, thereby still more preferably interacting with the gH/gLheterodimer, which still more preferably results in dislodging theprofusion domain of gD.

The recombinant herpesvirus of the present invention may, furthermore,encode one or more molecule(s) that stimulate(s) the host immuneresponse against a cell, preferably a diseased cell, as defined above. Amolecule that stimulates the host immune response is also termed“immunotherapy molecule”. Thus, the recombinant herpesvirus of thepresent invention may be a combined oncolytic and immunotherapeuticvirus. An immunotherapeutic virus is a virus that encodes molecules thatboost the host immune response to a cell, i.e. that stimulate the hostimmune response so as to be directed against a cell. An example of sucha virus is T-VEC (Liu et al., 2003).

Immunotherapy molecules, in addition to the chimeric gB, enable therecombinant virus, besides the specific targeting and killing of a cellvia the heterologous polypeptide ligand, to stimulate a subject's immunesystem in a specific or unspecific manner. Expression of immunotherapymolecules by the recombinant virus in a subject can induce an immuneresponse which finally results in the killing of diseased cells.Immunotherapy may act specifically wherein the immunotherapy moleculesstimulate the subject's immune system against one or some specificantigen(s) present on (a) cell(s). For example, an immunotherapymolecule may be an antibody which is directed against a specific cellsurface receptor, e.g.

CD20, CD274, and CD279. Once bound to an antigen, antibodies can induceantibody-dependent cell-mediated cytotoxicity, activate the complementsystem, or prevent a receptor from interacting with its ligand. All thatcan lead to cell death. Preferred cells are tumor cells. This techniqueis known and approved in the art. There are multiple antibodies whichare approved to treat cancer, including Alemtuzumab, Ipilimumab,Nivolumab, Ofatumumab, and Rituximab. Alternatively, the immunotherapymolecule can act non-specifically by stimulating the subject's immunesystem. Examples of immunotherapy molecules are inter alias cytokines,chemokines or immune checkpoint regulators. For example, some cytokineshave the ability to enhance anti-tumor activity and can be used aspassive cancer treatments. The use of cytokines as immunotherapymolecules is known in the art. Examples of cytokines are GM-CSF,interleukin-2, interleukin-12, or interferon-α. GM-CSF is used, forexample in the treatment of hormone-refractory prostate cancer orleukemia. Interleukin-2 is used, for example, in the treatment ofmalignant melanoma and renal cell carcinoma. IL-12 is used in theexperimental treatment of glioblastoma. Interferon-α is, for example,used in the treatment of hairy-cell leukemia, AIDS-related Kaposi'ssarcoma, follicular lymphoma, chronic myeloid leukemia and malignantmelanoma.

The recombinant herpesvirus of the present invention may be attenuated,for example by deletions in or alterations of genes known to attenuatevirus virulence, such as the viral genes γ₁34.5, UL39, and/or ICP47. Theterm “attenuated” refers to a weakened or less virulent herpesvirus.Preferred is a conditional attenuation, wherein the attenuation affectsonly non-diseased cells. More preferred, only the diseased cells such astumor cells are affected by the full virulence of the herpesvirus. Aconditional attenuation can be achieved, for example, by thesubstitution of the promoter region of the γ₁34.5, UL39 and/or ICP47gene with a promoter of a human gene that is exclusively expressed indiseased cells (e.g. the survivin promoter in tumor cells). Furthermodifications for a conditional attenuation may include the substitutionof regulatory regions responsible for the transcription of IE genes(immediate early genes) like the ICP-4 promoter region with promoterregions of genes exclusively expressed in diseased cells (e.g. thesurvivin promoter). This change will result in a replication conditionalHSV, which is able to replicate in diseased cells but not in normalcells. Additional modification of the virus may include the insertion ofsequence elements responsive to microRNAs (miRs), which are abundant innormal but not tumor cells, into the 3′ untranslated region of essentialHSV genes like ICP4. The result will be again a virus that isreplication incompetent only in normal cells.

In a second aspect, the present invention provides a pharmaceuticalcomposition comprising the recombinant herpesvirus of the presentinvention and a pharmaceutically acceptable carrier, optionallyadditionally comprising one or more molecule(s) that stimulate(s) thehost immune response against a cell, preferably a diseased cell, asdefined above. The recombinant herpesvirus of the present invention canbe used as a medicament. For the production of the medicament theherpesvirus has to be in a pharmaceutical dosage form comprising therecombinant herpesvirus of the present invention and a mixture ofingredients such as pharmaceutically acceptable carriers which providedesirable characteristics. The pharmaceutical composition comprises oneor more suitable pharmaceutically acceptable carrier which is/are knownto those skilled in the art.

The pharmaceutical composition may additionally comprise one or moremolecule(s) that stimulate(s) the host immune response against a cell.The definition of the one or more molecule(s) that stimulate(s) the hostimmune response against a cell is referred to above under the firstaspect of the present invention.

The pharmaceutical composition can be manufactured for systemic, nasal,parenteral, vaginal, topic, vaginal, intratumoral administration.Parental administration includes subcutaneous, intracutaneous,intramuscular, intravenous or intraperitoneal administration.

The pharmaceutical composition can be formulated as various dosage formsincluding solid dosage forms for oral administration such as capsules,tablets, pills, powders and granules, liquid dosage forms for oraladministration such as pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs, injectablepreparations, for example, sterile injectable aqueous or oleaginoussuspensions, compositions for rectal or vaginal administration,preferably suppositories, and dosage forms for topical or transdermaladministration such as ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the activity ofthe recombinant herpesvirus of the present invention, the dosage form,the age, body weight and sex of the subject, the duration of thetreatment and like factors well known in the medical arts.

The total dose of the compounds of this invention administered to asubject in single or in multiple doses may be in amounts, for example,from 10³ to 10¹⁰.

Single dose compositions may contain such amounts or submultiplesthereof to make up the daily dose. The dosages of the recombinantherpesvirus may be defined as the number of plaque forming unit (pfu).Examples of dosages include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰.

The recombinant herpesvirus of the present invention serves to treatdiseases in which diseased cells express specific target molecules ontheir surface, so that they are accessible from the outside of the cell,which target molecules are not produced by a normal cell or are producedby the normal cell to a lower degree. The normal cell may be therespective normal cell. “Respective” means that the diseased and normalcells are of the same origin, however, cells develop into diseased cellsdue to disease-generating influences, whereas other cells of same originremain healthy.

In a third aspect, the present invention provides the recombinantherpesvirus of the present invention, optionally in combination with oneor more molecule(s) that stimulate(s) the host immune response against acell, preferably a diseased cell, as defined above, for use in thetreatment of a tumor, infection, degenerative disorder orsenescence-associated disease. The recombinant herpesvirus of thepresent invention and the molecule that stimulates the host immuneresponse against a cell can be present within the same pharmaceuticalcomposition or within different pharmaceutical compositions. If they arepresent in different pharmaceutical compositions, they may beadministered simultaneously, or subsequently, either the herpesvirusbefore the molecule or the molecule before the herpesvirus. Theherpesvirus or the molecule may be administered at different frequenciesand/or time points. However, a combined treatment comprises that theherpesvirus and the molecule are administered at time intervals and/ortime points that allow the simultaneous treatment of the disease.

The present invention also discloses a method of treating a subjecthaving a tumor, infection, degenerative disorder orsenescence-associated disorder by administering a pharmaceuticallyeffective amount of the recombinant herpesvirus of the presentinvention.

The recombinant herpesvirus of the present invention may be administeredto a subject in combination with further treatments which stimulate thehost immune response against a cell, preferably a diseased cell, and/orserve to treat the specific disease of the subject. Such furthertreatments may include other drugs, chemotherapy, radiotherapy,immunotherapy, combined virotherapy, etc.

The present invention also discloses the use of the herpesvirus of thepresent invention, optionally in combination with one or moremolecule(s) that stimulate(s) the host immune response against a cell,preferably a diseased cell, as defined above, for the preparation of apharmaceutical composition for the treatment of a tumor, infection,degenerative disorder or senescence-associated disease.

The subjects which are treated by the recombinant herpesvirus of thepresent invention are preferably humans.

In a fourth aspect, the present invention provides a nucleic acidmolecule comprising a nucleic acid coding for the chimeric gB of thepresent invention having fused or inserted the ligand. The nucleic acidmolecule may be the genome of the recombinant herpesvirus of the presentinvention or a part thereof. Preferably, the nucleic acid moleculeencodes the precursor form of the chimeric gB including the signalsequence of the gB glycoprotein. If the chimeric gB was engineered toharbor the ligand to its N-terminal amino acid, the correspondingnucleic acid has the nucleic acid sequence of the ligand insertedbetween the last amino acid of the signal sequence and the first aminoacid of the mature protein.

In a fifth aspect, the present invention provides a vector comprisingthe nucleic acid molecule. Suitable vectors are known in the art andinclude plasmids, cosmids, artificial chromosomes (e.g. bacterial, yeastor human), bacteriophages, viral vectors (retroviruses, lentiviruses,adenoviruses, adeno-associated viruses), in particular baculovirusvector, or nano-engineered substances (e.g. ormosils). In oneembodiment, the vector is modified, in particular by a deletion,insertion and/or mutation of one or more nucleic acid bases, such thatits virulence is attenuated, preferably in case of a viral vector, orthat it replicates conditionally in diseased cells but not innon-diseased cells. For example, deletion of one or both copies of theγ₁34.5 gene, the UL39 gene, the ICP47 gene results in attenuation of thevirus. Attenuation or attenuated refers to weakened or less virulentvirus.

Moreover, the substitution of the promoter region of the γ₁34.5 genewith a promoter of a human gene that is exclusively expressed indiseased cells, e.g. tumor cells (e.g. survivin promoter in tumorcells), which will result in an attenuated phenotype in non-diseasedcells and non-attenuated phenotype in diseased cells, is included.Further modifications may include the substitution of regulatory regionsresponsible for the transcription of IE genes like the ICP-4 promoterregion with promoters of genes exclusively expressed in diseased cells(e.g. survivin promoter). This change will produce a replicationconditional herpesvirus, able to replicate in diseased cells but not innormal cells. Cell culture cells for propagation of the virus progenywill provide high levels of specific promoter activating proteins toallow for the production of high virus yields.

In a sixth aspect, the present invention provides a polypeptidecomprising the chimeric gB having fused or inserted the ligand.

In a seventh aspect, the present invention provides a cell comprisingthe recombinant herpesvirus, the nucleic acid molecule comprising anucleic acid coding for the chimeric gB of the present invention havingfused or inserted the ligand, the vector comprising the nucleic acidmolecule, or the polypeptide comprising the chimeric gB having fused orinserted the ligand. Preferably, the cell is a cell culture cell.Suitable cell cultures and culturing techniques are well known in theart (Peterson and Goyal, 1988).

In an eighth aspect, the present invention provides a method forinfecting a cell using the recombinant herpesvirus of the presentinvention. The object of the present invention is the provision of arecombinant herpesvirus which infects a cell unwanted in a subject,propagates therein, lyses the cell and, thereby, kills the cell. Themethod for infecting also serves for growth of the recombinantherpesvirus in a cell present in cell culture. “Infecting” means thatthe virus enters the cell via fusion of the viral surface membrane withthe cell membrane and viral components such as the viral genome arereleased into the cell. Methods of infecting a cell with a virus areknown in the art, e.g. by incubating the virus with the cell to beinfected (Florence et al., 1992; Peterson and Goyal, 1988). “Killing”means that the cell is totally eliminated due to the infection of theherpesvirus of the present invention, the production of viral particleswithin the cell and, finally, the release of the new viral particles bylysing the cell. Cells, for example in a cell culture, which carry thetarget molecule of the ligand on their surface can be used to test thelytic efficacy of the recombinant herpesvirus. For example, the cell maybe a diseased cell obtained from a subject, for example a tumor cell.This cell is infected and thereby killed by the recombinant herpesvirus.The successful killing of the cell is indicative of the cell specificityof the recombinant herpesvirus, in order to evaluate the therapeuticsuccess of eliminating cells such as tumor cells from the subject. In afurther embodiment, also non-diseased cells may be obtained from thesame subject or from a control subject not suffering from the disease,i.e. the cells do not carry the target molecule of the ligand on theirsurface or carry the target molecule to a lower extent. By this, it canbe tested whether and/or to which extent the non-diseased cell issusceptible to infection by the recombinant herpesvirus. In anotherembodiment, diseased cells comprised in a population of cells (e.g.tissue such as blood) comprising non-diseased cells and diseased cells(for example tumor cells such as leukemia cells) are killed afterisolation of the population of cells from a subject (e.g.leukapheresis). This serves to obtain a population of cells free ofdiseased cells, e.g. blood free of diseased cells such as leukemiacells, in particular for a later transplant of the population of cellsinto a subject, preferably into the same subject the population of cellswas isolated from.

In case of blood and leukemia, for example, this method provides forre-infusion of blood free of tumor cells. The method for killing a cellusing the recombinant herpesvirus of the present invention may be anin-vitro or in-vivo method.

In a ninth aspect, the present invention provides an in-vitro method forproducing a recombinant herpesvirus in a cell present in cell cultureusing the herpesvirus according to any one of claims 1 to 9, preferablywherein the cell expresses or binds as a target molecule an artificialmolecule, more preferably the target molecule comprises an antibody, anantibody derivative or an antibody mimetic, still more preferably anscFv, still more preferably an scFv capable of binding to a part of theGCN4 yeast transcription factor, still more preferably an scFv capableof binding to the part of the GCN4 yeast transcription factor ascomprised by SEQ ID NO: 37, still more preferably the scFv as comprisedby SEQ ID NO: 39, most preferably the molecule identified by thesequence of SEQ ID NO: 41.

The recombinant herpesvirus of the present invention serves the purposeof infecting and killing diseased cells in humans. This requires theprovision of the herpesvirus and, therefore, its propagation andproduction. As propagation of the herpesvirus shall be avoided indiseased cells, so as to avoid the introduction of material such as DNA,RNA and/or protein of diseased cells such as tumor cells in humans, therecombinant herpesvirus has to be engineered to be capable of infectingalso non-diseased cells. This requires the retargeting of therecombinant herpesvirus to diseased cells for killing and tonon-diseased cells for propagation. Therefore, the ninth aspect of thepresent invention comprises the modification of the recombinantherpesvirus with more than one, such as 2, 3 or 4, preferably 2,heterologous polypeptide ligands. The ligands may be comprised by gBonly, but may also be comprised by gB and gD and optionally by gH.

Consequently, in an embodiment of the ninth aspect, the recombinantherpesvirus comprises a heterologous polypeptide ligand, fused to orinserted into gB, capable of binding to a target molecule present on thecell present in cell culture and an additional heterologous polypeptideligand fused to or inserted into gB, gD and/or gH, capable of binding toa target molecule present on a diseased cell. Preferably, the chimericgB comprises a heterologous polypeptide ligand capable of binding to atarget molecule present on the cell present in cell culture and amodified gD and/or gH comprise(s) a heterologous polypeptide ligandcapable of binding to the target molecule present on a diseased cell.

Suitable techniques and conditions for growing herpesvirus in a cell arewell known in the art (Florence et al., 1992; Peterson and Goyal, 1988)and include incubating the herpesvirus with the cell and recovering theherpesvirus from the medium of the infected cell culture. The cell bywhich the recombinant herpesvirus is produced carries a target moleculeto which the recombinant herpesvirus binds via the heterologouspolypeptide ligand. Preferably, the target molecule is an artificialtarget molecule. The artificial target molecule is specificallyconstructed to bind to the heterologous polypeptide ligand. Conversely,the ligand is specifically selected and constructed to bind to theartificial target molecule. Thus, the target molecule may be an antibodywhich is not naturally produced by the target cell, an antibodyderivative or an antibody mimetic, preferably an scFv. The heterologouspolypeptide ligand may be a natural polypeptide, preferably a fungal orbacterial polypeptide, such as a polypeptide from the genusSaccharomyces such as Saccharomyces cerevisiae, or an artificialpolypeptide such as a part of the natural polypeptide capable of bindingto the target molecule. The cell may be any cultured cell which issuitable for growth of herpesvirus. Preferably, the cell is anon-diseased cell. The cell may be present as a cell line or may be anisolated cell, preferably the cell is present as a cell line. The cellline may be approved for herpesvirus growth. Suitable cell lines areVero, 293, 293T, HEp-2, HeLa, BHK, or RS cells, most preferably a Verocell.

A “cultured” cell is a cell which is present in an in vitro cell culturewhich is maintained and propagated, as known in the art. Cultured cellsare grown under controlled conditions, generally outside of theirnatural environment. Usually, cultured cells are derived frommulticellular eukaryotes, especially animal cells. “A cell line approvedfor growth of herpesvirus” is meant to include any cell line which hasbeen already shown that it can be infected by a herpesvirus, i. e. thevirus enters the cell, and is able to propagate and produce the virus. Acell line is a population of cells descended from a single cell andcontaining the same genetic composition. Preferred cells for propagationand production of the recombinant herpesvirus are Vero, 293, 293T,HEp-2, HeLa, BHK, or RS cells.

In a preferred embodiment of the in-vitro method, the ligand is a partof a natural polypeptide and preferably has a length of 274 amino acidresidues or less, preferably of less than 200 amino acid residues, morepreferably of less than 50 amino acid residues, still more preferablyless than 40 amino acids residues, and still more preferably of betweent 10 and 30 amino acids such as 20 amino acids, whereby the part allowsthe construction of target molecules and the retargeting of theherpesvirus to a cell carrying the respective target molecule. Thetarget molecule is an antibody derivative capable of binding to the partof the natural polypeptide. More preferably, the heterologouspolypeptide ligand is a part of the GCN4 yeast transcription factor, thetarget molecule is an antibody derivative capable of binding to theligand and the cell is a cell which has been modified to express thetarget molecule. Most preferably, the heterologous polypeptide ligand isthe molecule identified by the sequence of SEQ ID NO: 37, the targetmolecule is the molecule identified by the sequence of SEQ ID NO: 41 andthe cell is the Vero cell line which has been modified to express themolecule identified by the sequence of SEQ ID NO: 41, herein named VeroGCN4 cell line.

The Vero-GCN4 cell line expresses an artificial receptor being an scFvto the GCN4 peptide. The Vero-GCN4 cell line serves the purpose ofenabling the cultivation of herpesvirus recombinants retargeted toHER2-positive cells, and detargeted from natural herpesvirus receptors.Because HER2 is an oncogene, and the HER2-positive cells are cancercells, growth and production of oncolytic recombinant herpesvirusdestined to human use in cancer cells should be avoided, in order toavoid the possible, accidental introduction of tumor-derived material(DNA, RNA, proteins) in humans. At the same time, the herpesvirus shouldbe capable of infecting diseased cells. Therefore, the Vero-GCN4 cellline and an HER2-retargeted herpesvirus were constructed. The Vero-GCN4cell line expresses an artificial receptor made of an scFv to the GCN4peptide, fused to extracellular domains 2 and 3, transmembrane (TM) andC-tail of Nectin-1. The HER2 retargeted herpesvirus expresses the GCN4peptide in gB. Consequently, the recombinant herpesvirus issimultaneously retargeted to HER2, in order to infect cancer cells, andto the GCN4 peptide, in order to infect the Vero-GCN4 cell line forvirus growth and production.

In a particularly preferred embodiment of the ninth aspect, gB comprisesa ligand capable of binding to a target molecule present on a cellpresent in cell culture, whereby the ligand may be an artificialpolypeptide, more preferably a part of a natural polypeptide, and stillmore preferably a part of the GCN4 yeast transcription factor, and amodified gD and/or modified gH comprise(s) a ligand capable of bindingto a target molecule present on a diseased cell, whereby the targetmolecule may be an antibody, an antibody derivative or an antibodymimetic, still more preferably an scFv, and still more preferably anscFv capable of binding to HER2. In the most preferred embodiment, therecombinant herpesvirus comprises a chimeric gB comprising the moleculeidentified by the sequence of SEQ ID NO: 37 and a modified gD and/or gHcomprise(s) an scFv identified by SEQ ID NO: 32.

Such herpesvirus is capable of infecting the Vero-GCN4 cell lineexpressing the molecule identified by the sequence of SEQ ID NO: 41 forpropagation and of infecting a tumor cell through HER2 present on thetumor cell for killing the tumor cell.

In another particularly preferred and most preferred embodiment, gBcomprises a ligand capable of binding to a target molecule present on adiseased cell, and gD comprises a ligand capable of binding to a targetmolecule present on a cell present in cell culture. The definitions ofligand, target molecule and cell are as in the preceding chapter.

FIGURES

FIG. 1: Genome arrangements of recombinants R-BP901, R-BP903, R-BP909,R-313, R-315, R-317 and R-319. (A-G) The HSV-1 genome is represented asa line bracketed by internal repeats (IR). The Lox-P-bracketed BACsequence and eGFP fluorescent marker are inserted in the intergenicregion U_(L)3-U_(L)4. (A) R-BP903 carries the insertion of scFv-HER2,with a downstream 12 Ser-Gly linker, between AA 43-44 of gB. (B) R-BP909is the same as R-BP903, but, in addition carries the deletion of AA 6-38from mature gD for detargeting purpose. (C) R-BP901 carries theinsertion of scFv-HER2, with a Ser-Gly linkers, between AA 81-82 of gB.(D) R-313 carries the insertion of GCN4 peptide, with one upstream andone downstream Ser-Gly linker, between AA 43-44 of immature gB and thescFv-HER2, with a downstream Ser-Gly, 11 amino acid long linker, inplace of AA 6-38 of mature gD. E) R-315 carries the insertion of GCN4peptide, with one upstream and one downstream Ser-Gly linker, between AA81-82 of immature gB and the scFv-HER2, with a downstream Ser-Gly, 11amino acid long linker, in place of AA 6-38 of mature gD. F) R-317carries the insertion of GCN4 peptide, with one upstream and onedownstream Ser-Gly linker, between AA 76-77 of immature gB and thescFv-HER2, with a downstream Ser-Gly, 11 amino acid long linker, inplace of AA 6-38 of mature gD. G) R-319 carries the insertion of GCN4peptide, with one upstream and one downstream Ser-Gly linker, between AA95-96 of immature gB and the scFv-HER2, with a downstream Ser-Gly, 11amino acid long linker, in place of AA 6-38 of mature gD.

FIG. 2: R-BP901 and R-BP909 express the chimeric scFv-gB glycoprotein.Lysates of SK-OV-3 cells infected with R-BP901, R-BP909 or R-LM5, at aninput multiplicity of infection of 3 PFU/cell were subjected to PAGE. gBwas detected by immunoblot with MAb H1817. Numbers on the left representthe migration position of the 250 K, 130 K and 95 K MW markers.

FIG. 3: Infection of J cells expressing single receptors withrecombinants R-BP901, R-BP903 and R-BP909. J cells express no receptorfor wt-HSV. J-HER2, J-Nectin1, J-HVEM only express the indicatedreceptor. The indicated cells were infected with R-BP903, R-BP909 andR-BP901 and monitored for green fluorescence microscopy 24 h postinfection. (A) R-BP903 infected J-HER2 cells, as well as J-Nectin andJ-HVEM, as expected given that this recombinant encodes a wt-gD. Thisvirus is retargeted to HER2 and retains the natural tropism. (B) R-BP909infects cells that express HER2 as the sole receptor (J-HER2) and failsto infect J-Nectin and J-HVEM, as a consequence of gD deletion of AA6-38. R-BP909 is retargeted to HER2 and detargeted from HSV-1 gD naturalreceptors. (C) R-BP901 fails to infect J-HER2; this virus is notretargeted to HER2.

FIG. 4: R-BP909 specifically infects HER2⁺ cancer cells. The indicatedHER2⁻ and HER2⁺ cancer cell lines were infected with R-BP909 andR-BP903. Pictures were taken 24 h after infection at fluorescencemicroscope. R-BP909 infects the HER2-positive cancers cells and fails toinfect the HER2-negative cancer cells. R-BP903 infects cellsirrespective of the expression of HER2, in agreement with the lack ofdetargeting.

FIG. 5: Characterization of R-BP909 entry pathways in J-HER2 (A) andSK-OV-3 (B) cells. The indicated viruses were preincubated with HD1,52S, H126 MAb and then allowed to infect J-HER2 or SK-OV-3 cells. Whenindicated, cells were pretreated with trastuzumab or control IgGs.Infection was quantified 24 h later by means of flow cytometry. (A)R-BP909 infection of J-HER2 cells is almost abolished by trastuzumab,and by MAb H126 to gB. (B) R-BP909 infection of SK-OV-3 cells isinhibited by trastuzumab and by MAb H126 to gB, 52S to gH, but not byMAb HD1 to gD. R-LM113, a recombinant retargeted to HER2 throughinsertion of scFv to HER2 in gD, behaved similarly to R-BP909.

FIG. 6: Growth curves of R-BP909, and of the control recombinantsR-VG809 (retargeted to HER2 through gH) and R-LM113 (retargeted to HER2through gD). SK-OV-3 cells were infected with the indicated recombinantsat an input multiplicity of infection of 0.1 PFU/cell and harvested atthe indicated times (h) after infection. Progeny virus was titrated inSK-OV-3 cells. Growth curves indicate that R-BP909 replicated in asimilar way to R-VG809, about one log less than R-LM113.

FIG. 7: Killing ability of R-BP909 and R-VG809 for SK-OV-3 andMDA-MB-453 cells infected, and lack of killing ability for HER2⁻ cancercells. The HER2-positive SK-OV-3 and MDA-MB-453 cells, and the HER2⁻MDA-MB-231 cancer cells were infected with the indicated viruses at 2PFU/cell (0.1 PFU/cell for MDA-MD-231 cells), respectively. Viabilitywas quantified by AlamarBlue assay. R-BP909 killed the SK-OV-3 andMDA-MB-453 cells with similar efficiency to R-VG809. Both viruses failedto kill the HER2⁻ negative MDA-MD-231 cancer cells, consistent withtheir inability to infect these cells.

FIG. 8: Pattern of infection of the recombinants R-313, R-315, R-317 andR-319. wt-Vero, Vero-GCN4R, SK-OV-3, parental J and J cells that expressreceptors for wt-HSV J-HVEM and J-Nectin were infected with theindicated viruses and monitored for green fluorescence microscopy 24 hpost infection. R-313, R-315, R-317 and R-319 infect cells that expressHER2 (both human and simian) and GCN4 as receptors and fails to infectthrough Nectin and HVEM, as a consequence of gD deletion of AA 6-38. Allthe engineered viruses are retargeted to HER2 and GCN4 and detargetedfrom HSV-1 gD natural receptors. Inhibition of infection inHER2-positive cell lines exposed to Trastuzumab (alias Herceptin)confirms that R-313, R-315, R-317 and R-319 employ the HER2 as theportal of entry in these cells.

FIG. 9: Growth curves of R-313, R-315, R-317, R-319 and of the controlrecombinants R-LM113 (retargeted to HER-2 through gD) and R-LM5 (wt forHSV glycoproteins and with other genomic modifications present in R-313,R-315, R-317, R-319 and R-LM113). Vero-GCN4R and SK-OV-3 cells wereinfected with the indicated recombinants at an input multiplicity ofinfection of 0.1 PFU/cell (as titrated in the respective cell lines) andharvested at the indicated times (h) after infection. Progeny virus wastitrated in SK-OV-3 cells.

FIG. 10: Plating efficiency of R-313, R-315, R-317, R-319 and of thecontrol recombinants R-LM113 and R-LM5 in different cell lines.Replicate aliquots of the recombinant viruses were plated ontoVero-GCN4R, wt-Vero and SK-OV-3. At 3 days after infection, plaques werescored under the fluorescence microscope.

FIG. 11: Relative plaque size of R-313, R-315, R-317 and R-319 indifferent cell lines. A) Replicate aliquots of R-313, R-315, R-317,R-319, R-LM113 and R-LM5 were plated in Vero-GCN4R, wt-Vero and SK-OV-3.Pictures of plaques were taken at the fluorescence microscope 3 daysafter infection. Representative plaques are shown. B) Quantification ofplaque areas is shown pxE2.

FIG. 12: Schematic drawing of the chimeric scFv to GCN4-Nectin receptor.The receptor presents N-terminal leader peptide and HA tag sequence,followed by the scFv to GCN4, placed between two short linker, GA andGSGA linker. The second part of the molecule corresponds to humanNectin-1 (PVRL1) residues Met143 to Val517 comprising the Nectin-1extracellular domains 2 and 3, the TM segment and the intracellularcytoplasmic tail.

FIG. 13: Stability of Vero-GCN4 positive cells. The expression of thescFv GCN4-Nectin receptor was analysed by FACS by means of Mab to HAtag.

Diagrams show the percentage positive cells from Vero GCN4 clone 11.2cells at passages 10, 15, 30, 40. Result: the expression of theartificial receptor remained stable after 40 consecutive passages.

FIG. 14: Genome arrangement of the recombinant R-321. The HSV-1 genomeis represented as a line bracketed by internal repeats (IR). TheLox-P-bracketed BAC sequence and eGFP fluorescent marker are inserted inthe intergenic region U_(L)3-U_(L)4. R-321, carries the deletion of AA30 and 38 of mature gD and the insertion of scFv-HER2 after AA 37 of gD.R-321 carries the insertion of GCN4 peptide, with one upstream and onedownstream Ser-Gly linker, between AA 43-44 of immature gB.

FIG. 15: Pattern of infection of the recombinant R-321. wt-Vero,Vero-GCN4R, SK-OV-3, parental J and J cells that express receptors forwt-HSV J-HVEM and J-Nectin were infected with the indicated viruses andmonitored for green fluorescence microscopy 24 h post infection. R-321infects cells that express HER2 (both human and simian) and GCN4 asreceptors and fails to infect through Nectin and HVEM, as a consequenceof gD deletion of AA 30 and 38. R-321 is retargeted to HER2 and GCN4 anddetargeted from HSV-1 gD natural receptors. Inhibition of infection inHER2-positive cell lines exposed to Trastuzumab (alias Herceptin)confirms that R-321 employs the HER2 as the portal of entry in thesecells.

FIG. 16: Growth curves of R-321 and of the control recombinants R-LM113(retargeted to HER-2 through gD) and R-LM5 (wt for HSV glycoproteins andwith other genomic modifications present in R-321). Vero-GCN4R andSK-OV-3 cells were infected with the indicated recombinants at an inputmultiplicity of infection of 0.1 PFU/cell (as titrated in thecorrespondent cell lines) and harvested at the indicated times (h) afterinfection. Progeny virus was titrated in SK-OV-3 cells.

SEQUENCES

SEQ ID NO: 1: amino acid sequence of HSV-1 gB wild type, precursor(Human herpesvirus 1 strain F, GenBank accession number: GU734771.1; gBencoded by positions 52996 to 55710).

SEQ ID NO: 2: amino acid sequence of the precursor of gB (SEQ ID NO: 1)having inserted the trastuzumab scFv between amino acids 43 and 44, asencoded by constructs R-BP903 and R-BP909. Linker SSGGGSGSGGSG (SEQ IDNO: 30) is introduced between the C-terminal amino acid sequence of thescFV insert and amino acid 44 of gB.

SEQ ID NO: 3: amino acid sequence of the precursor of gB (SEQ ID NO: 1)having inserted the trastuzumab scFv between amino acids 81 and 82, asencoded by construct R-BP901. Linker HSSGGGSG (SEQ ID NO: 29) isintroduced between amino acid 81 of gB and the N-terminal amino acidsequence of the scFV insert. Linker SSGGGSGSGGSG (SEQ ID NO: 30) isintroduced between the C-terminal amino acid sequence of the scFV insertand amino acid 82 of gB.

SEQ ID NO: 4: amino acid sequence of HSV-1 gD wild type, precursor(Human herpesvirus 1 strain F, GenBank accession ID: GU734771.1; gDencoded by positions 138281 to 139465).

SEQ ID NO: 5: amino acid sequence of HSV-1 gD wild type, precursor (SEQID NO: 4), with deletion of amino acids 6 to 38 of mature gD, as encodedby R-BP909.

SEQ ID NO: 6: amino acid sequence of HSV-1 deleted gD (SEQ ID NO: 5),having inserted the trastuzumab scFv between amino acids 30 and 64, asencoded by construct R-LM113. Amino acids EN were introduced to insert arestriction site for easiness of engineering and screening.

SEQ ID NO: 7: Trastuzumab scFv cassette bracketed by Ser-Gly linkers,present in plasmid named pSG-scFvHER2-SG, as in R-BP901, encoding theinsert in SEQ ID NO: 3.

SEQ ID NO: 8: amino acid sequence encoded by SEQ ID NO: 7; amino acids 1to 8 are the upstream Ser-Gly linker (SEQ ID NO: 29), amino acids 9 to116 are the V_(L) region, amino acids 117 to 136 is the linker thatconnects the V_(L) and V_(H) regions (SEQ ID NO: 31), amino acids 137 to255 encode the V_(H) region, amino acids 256 to 267 encode thedownstream 12 Ser-Gly linker (SEQ ID NO: 30).

SEQ ID NO: 9: The Trastuzumab scFv cassette, present in plasmid namedp-SG-scFvHER2-SG, but lacking the 8 residues long upstream Ser-Glylinker in R-BP903 and R-BP909, encoding the insert in SEQ ID NO: 2.

SEQ ID NO: 10: amino acid sequence encoded by SEQ ID NO: 9; amino acids1 to 108 are the V_(L) region, amino acids 109 to 128 is the linker thatconnects the V_(L) and V_(H) regions (SEQ ID NO: 31), amino acids 129 to247 encode the V_(H) region, amino acids 248 to 259 encode thedownstream 12 Ser-Gly linker (SEQ ID NO: 30).

SEQ ID NO: 11: gB43GalKfor

SEQ ID NO: 12: gB43GalKrev

SEQ ID NO: 13: gB43_sc4D5_for

SEQ ID NO: 14: gB43_sc4D5_rev

SEQ ID NO: 15: gB81fGALK

SEQ ID NO: 16: gB81GALKrev

SEQ ID NO: 17: gB81sc4D5f

SEQ ID NO: 18: gB81SGr

SEQ ID NO: 19: scFv4D5 358 r

SEQ ID NO: 20: scFv4D5 315 f

SEQ ID NO: 21: gD5_galK_f

SEQ ID NO: 22: gD39_galK_r

SEQ ID NO: 23: gD_aa5_39_f

SEQ ID NO: 24: gD_aa5_39_r

SEQ ID NO: 25: galK_129_f

SEQ ID NO: 26: galK_417_r

SEQ ID NO: 27: gB_ext_for

SEQ ID NO: 28: gB_431_rev

SEQ ID NO: 29: 8 Ser-Gly linker

SEQ ID NO: 30: 12 Ser-Gly linker

SEQ ID NO: 31: Linker connecting V_(L) and V_(H) regions

SEQ ID NO: 32: Trastuzumab scFv

SEQ ID NO: 33: GCN4gB_43_44_JB

SEQ ID NO: 34: GCN4gB_43_44_rB

SEQ ID NO: 35: amino acid sequence of the precursor of gB (SEQ ID NO: 1)having inserted the GCN4 peptide between amino acids 43 and 44, asencoded by the construct R-313. The GCN4 peptide is flanked by a Ser-Glylinker.

SEQ ID NO: 36: nucleotide sequence encoding GCN4 peptide with upstreamand downstream linkers for recombination into gB

SEQ ID NO: 37: GCN4 peptide

SEQ ID NO: 38: GCN4 epitope

SEQ ID NO: 39: amino acid sequence of scFv to GCN4 peptide

SEQ ID NO: 40: nucleotide sequence encoding scFv-GCN4-Nectin1 chimera

SEQ ID NO: 41: amino acid sequence encoded by SEQ ID NO: 40; amino acidsequence of the scFv capable of binding to the GCN4 peptide comprisingan N-terminal leader peptide, an HA tag sequence, a short GA linker, thescFv sequence from amino acids 33 to 275, a short GSGA linker, and humanNectin-1 (PVRL1) residues Met143 to Val517

SEQ ID NO: 42: Genbank accession number AJ585687.1 (gene encoding theGCN4 yeast transcription factor

SEQ ID NO: 43: amino acid sequence of the GCN4 yeast transcriptionfactor UniProtKB—P03069 (GCN_YEAST)

SEQ ID NO: 44: gB_76_galK_for

SEQ ID NO: 45: gB_76_galK_rev

SEQ ID NO: 46: gB_76_GCN4_for

SEQ ID NO: 47: gB_76_GCN4_rev

SEQ ID NO: 48: amino acid sequence of the precursor of gB (SEQ ID NO: 1)having inserted the GCN4 peptide between amino acids 76 and 77, asencoded by the construct R-317. The GCN4 peptide is flanked by a Ser-Glylinker.

SEQ ID NO: 49: gB_81_GCN4_for

SEQ ID NO: 50: gB_81_GCN4_rev

SEQ ID NO: 51: amino acid sequence of the precursor of gB (SEQ ID NO: 1)having inserted the GCN4 peptide between amino acids 81 and 82, asencoded by the construct R-315. The GCN4 peptide is flanked by a Ser-Glylinker.

SEQ ID NO: 52: gB_95_galK_for

SEQ ID NO: 53: gB_95_galK_rev

SEQ ID NO: 54: gB_95_GCN4_for

SEQ ID NO: 55: gB_95_GCN4_rev

SEQ ID NO: 56: amino acid sequence of the precursor of gB (SEQ ID NO: 1)having inserted the GCN4 peptide between amino acids 95 and 96, asencoded by the construct R-319. The GCN4 peptide is flanked by a Ser-Glylinker.

SEQ ID NO: 57: gD5_galK_f

SEQ ID NO: 58: scFv_galK_rev

SEQ ID NO: 59: gDdel30_38 for

SEQ ID NO: 60: gDdel30_38 rev

SEQ ID NO: 61: amino acid sequence of the precursor of gD (SEQ ID NO: 4)having deleted amino acids 30 and 38 and inserted the trastuzumab scFvafter amino acid 37 with regard to mature gD, as encoded by theconstruct R-321.

SEQ ID NO: 62: amino acid sequence of HSV-1 gD wild type, mature form(Human herpesvirus 1 strain F, GenBank accession ID: GU734771.1).

Examples Example 1:

Construction of HSV Recombinants Expressing Genetically Modified gBsCarrying a Single Chain Antibody (scFv) Directed to HER2 (scFv-HER2)(R-BP901, R-BP903, R-BP909), without or with Deletion in the gD HSVGene, and Encoding eGFP as Reporter Gene, or Carrying the GCN4 Peptide(R-313)

A) R-BP903: insertion of scFv-HER2 between AA (amino acid) 43 and 44 ofHSV gB.

The inventors engineered R-BP903—this clone has also the nameR-903—(FIG. 1A) by insertion of the sequence encoding the trastuzumabscFv between AA 43 and 44 of immature gB, corresponding to AA 13 and 14of mature gB, after cleavage of the signal sequence, which encompassesAA 1-30. The starting genome was the BAC LM55, which carriesLOX-P-bracketed pBeloBAC11 and eGFP sequences inserted between UL3 andUL4 of HSV-1 genome (Menotti et al., 2008). The engineering wasperformed by means of galK recombineering. Briefly, the galK cassettewith homology arms to gB was amplified by means of primers gB43GalKforGGTGGCGTCGGCGGCTCCGAGTTCCCCCGGCACGCCTGGGGTCGCGGCCGCGCCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 11) and gB43GalKrevGGCCAGGGGCGGGCGGCGCCGGAGTGGCAGGTCCCCCGTTCGCCGCCTGGGTTCAGCACTGTCCTGCTCCTT (SEQ ID NO: 12) using pGalK as template. Thiscassette was electroporated in SW102 bacteria carrying LM55 BAC. Therecombinant clones carrying the galK cassette were selected on platescontaining M63 medium (15 mM (NH₄)₂50₄, 100 mM KH₂PO₄, 1.8 μg FeSO₄.H₂O,adjusted to pH7) supplemented with 1 mg/L D-biotin, 0.2% galactose, 45mg/L L-leucine, 1 mM MgSO₄.7H₂O and 12 μg/ml chloramphenicol. In orderto exclude galK false positive bacterial colonies, they were streakedalso on MacConkey agar base plates supplemented with 1% galactose and 12μg/ml chloramphenicol and checked by colony PCR with primer galK_129_fACAATCTCTGTTTGCCAACGCATTTGG (SEQ ID NO: 25) and galK_417_rCATTGCCGCTGATCACCATGTCCACGC (SEQ ID NO: 26). Next, the trastuzumab scFvcassette with the downstream Ser-Gly linker described below (SEQ ID NO:9; encoding SEQ ID NO: 10) and bracketed by homology arms to gB wasgenerated through the annealing and extension of primers gB43_sc4 D5_forGGTGGCGTCGGCGGCTCCGAGTTCCCCCGGCACGCCTGGGGTCGCGGCCGCGTCCGATATCCAGATGACCCAGTCCCCG (SEQ ID NO: 13) and gB43_sc4 D5_revGGCCAGGGGCGGGCGGCGCCGGAGTGGCAGGTCCCCCGTTCGCCGCCTGGGTACCGGATCCACCGGAACCAGAGCC (SEQ ID NO: 14). The recombinant genomeencodes for the chimeric gB, which carries the scFv to HER2 and onedownstream Ser-Gly linker, with sequence SSGGGSGSGGSG (SEQ ID NO: 30),and one linker between VL and VH region with the sequenceSDMPMADPNRFRGKNLVFHS (SEQ ID NO: 31). The recombinant clones carryingthe excision of the galK cassette and the insertion of the sequence ofchoice, scFv-HER2, were selected on plates containing M63 medium (seeabove) supplemented with 1 mg/L D-biotin, 0.2% deoxy-2-galactose, 0.2%glycerol, 45 mg/L L-leucine, 1 mM MgSO₄.7H₂O and 12 μg/mlchloramphenicol. Bacterial colonies were also checked for the presenceof sequence of choice by means of colony PCR with primers gB_ext_forGAGCGCCCCCGACGGCTGTATCG (SEQ ID NO: 27) and gB_431_revTTGAAGACCACCGCGATGCCCT (SEQ ID NO: 28).

B) R-BP909 (FIG. 1B): deletion of AA 6-38 from mature gD of R-BP903.R-BP909—this clone has also the name R-909—is identical to R-BP903 and,in addition, it carries the deletion of the sequence corresponding to AA6-38 in gD. The starting material was the R-BP903 BAC genome. Togenerate the AA 6-38 deletion in gD, galK cassette flanked by homologyarms to gD was amplified with primers gD5_galK_fTTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGCCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 21) and gD39_galK_rATCGGGAGGCTGGGGGGCTGGAACGGGTCCGGTAGGCCCGCCTGGATGTG TCAGCACTGTCCTGCTCCTT(SEQ ID NO: 22). Next, the inventors replaced galK sequence with asynthetic double-stranded oligonucleotide made of gD_aa5_39 fTTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGCACATCCAGGCGGGCCTACCGGACCCGTTCCAGCCCCCCAGCCTCCCGAT (SEQ ID NO: 23) and ofgD_aa5_39 r ATCGGGAGGCTGGGGGGCTGGAACGGGTCCGGTAGGCCCGCCTGGATGTGCGCCAAGGCATATTTGCCGCGGACCCCATGGAGGCCCACTATGACGACAA (SEQ ID NO: 24).

C) R-BP901—this clone has also the name R-901—(FIG. 1C): insertion ofscFv-HER2 between aa 81 and 82 of HSV gB.

The procedure was the same as described above to engineer the scFv-HER2in gB of R-BP903, with the following differences. First, the galKcassette was amplified by means of primers gB81fGALKCGGGGGACACGAAACCGAAGAAGAACAAAAAACCGAAAAACCCACCGCCGCCGCCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 15) and gB81GALKrevCGCAGGGTGGCGTGGCCCGCGGCGACGGTCGCGTTGTCGCCGGCGGGGC GTCAGCACTGTCCTGCTCCTT(SEQ ID NO: 16). Next, the trastuzumab scFv cassette bracketed by theSer-Gly linkers described below and by homology arms to gB was amplifiedas two separate fragments, named fragment #1 and fragment #2, frompSG-ScFvHER2-SG. pSG-ScFvHER2-SG carries a trastuzumab scFv cassettebracketed by Ser-Gly linkers (SEQ ID NO: 7, encoding SEQ ID NO: 8).Fragment #1 was amplified by means of primers gB81sc4D5fCGGGGGACACGAAACCGAAGAAGAACAAAAAACCGAAAAACCCACCGCCGCCGCATAGTAGTGGCGGTGGCTCTGGATCCG (SEQ ID NO: 17) and scFv4D5_358_rGGAAACGGTTCGGATCAGCCATCGG (SEQ ID NO: 19), using p-SG-ScFv-HER2-SG astemplate. Fragment#2 was amplified by means of primers gB81SGrCGCAGGGTGGCGTGGCCCGCGGCGACGGTCGCGTTGTCGCCGGCGGGGCGACCGGATCCACCGGAACCAGAGCC (SEQ ID NO: 18) and scFv4D5_315_fGGAGATCAAATCGGATATGCCGATGG (SEQ ID NO: 20) using pSG-ScFvHER2-SG astemplate. Fragments #1 and #2 were annealed and extended to generate thescFv-HER2 cassette, bracketed by the Ser-Gly linkers and the homologyarms to gB. The recombinant genome carries the scFv to HER2 bracketed byan upstream Ser-Gly linker, with sequence HSSGGGSG (SEQ ID NO: 29), anda downstream Ser-Gly linker, with sequence SSGGGSGSGGSG (SEQ ID NO: 30).The linker between VL and VH is SDMPMADPNRFRGKNLVFHS (SEQ ID NO: 31).

D) R-313: insertion of GCN4 peptide between AA 43 and 44 of HSV gB inHSV recombinant already expressing a scFv-HER2 in the deletion of AA6-38 in gD.

The inventors engineered R-313 (FIG. 1D) by insertion of the sequenceencoding the GCN4 peptide between AA 43 and 44 of immature gB,corresponding to AA 13 and 14 of mature gB after cleavage of the signalsequence, which encompasses AA 1-30. The starting genome was the BACLM113, which carries scFv-HER2 in place of AA 6 to 38 of gD,LOX-P-bracketed pBeloBAC11 and eGFP sequences inserted between U_(L)3and U_(L)4 of HSV-1 genome (Menotti et al., 2008). The engineering wasperformed by means of galK recombineering. Briefly, in order to insertthe GCN4 peptide in gB, the galK cassette with homology arms to gB wasamplified by means of primers gB43GalKforGGTGGCGTCGGCGGCTCCGAGTTCCCCCGGCACGCCTGGGGTCGCGGCCGCGCCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 11) and gB43GalKrevGGCCAGGGGCGGGCGGCGCCGGAGTGGCAGGTCCCCCGTTCGCCGCCTGGGTTCAGCACTGTCCTGCTCCTT (SEQ ID NO: 12) using pGalK as template. Thiscassette was electroporated in SW102 bacteria carrying the BAC LM 113BG. The recombinant clones carrying the galK cassette were selected onplates containing M63 medium (15 mM (NH₄)₂50₄, 100 mM KH₂PO₄, 1.8 μgFeSO₄.H₂O, adjusted to pH7) supplemented with 1 mg/L D-biotin, 0.2%galactose, 45 mg/L L-leucine, 1 mM MgSO₄.7H₂O and 12 μg/mlchloramphenicol. In order to exclude galK false positive bacterialcolonies, they were streaked also on MacConkey agar base platessupplemented with 1% galactose and 12 μg/ml chloramphenicol and checkedby colony PCR with primer galK_129_f ACAATCTCTGTTTGCCAACGCATTTGG (SEQ IDNO: 25) and galK_417_r CATTGCCGCTGATCACCATGTCCACGC (SEQ ID NO: 26).Next, the GCN4 peptide cassette (SEQ ID NO: 36, encoding SEQ ID NO: 37)with the downstream and upstream Ser-Gly linkers and bracketed byhomology arms to gB was generated through the annealing and extension ofprimers GCN4gB_43_44 JBGGTGGCGTCGGCGGCTCCGAGTTCCCCCGGCACGCCTGGGGTCGCGGCCGCGGGATCCAAGAACTACCACCTGGAGAACGAGGTGGCCAGACTGAAGAAGC TGGTGGGCAGC (SEQ IDNO: 33) and GCN4gB_43_44_rBGGCCAGGGGCGGGCGGCGCCGGAGTGGCAGGTCCCCCGTTCGCCGCCTGGGTGCTGCCCACCAGCTTCTTCAGTCTGGCCACCTCGTTCTCCAGGTGGTAG TTCTTGGATCC (SEQ IDNO: 34) which introduce a silent restriction site for the BamHIendonuclease, useful for screening of colonies by means of restrictionanalysis. The recombinant genome encodes the chimeric gB (SEQ ID NO:35), which carries the GCN4 peptide including one downstream and oneupstream Ser-Gly linker with the sequence GS. The recombinant clonescarrying the excision of the galK cassette and the insertion of thesequence of choice, GCN4 peptide, were selected on plates containing M63medium (see above) supplemented with 1 mg/L D-biotin, 0.2%deoxy-2-galactose, 0.2% glycerol, 45 mg/L L-leucine, 1 mM MgSO4.7H₂O and12 μg/ml chloramphenicol. Bacterial colonies were checked for thepresence of sequence of choice by means of colony PCR with primersgB_ext_for GAGCGCCCCCGACGGCTGTATCG (SEQ ID NO: 27) and gB_431_revTTGAAGACCACCGCGATGCCCT (SEQ ID NO: 28).

To reconstitute the recombinant virus R-BP909, 500 ng of recombinant BACDNA was transfected into the gD-complementing cell line named R6 (rabbitskin cell line expressing wt-gD under the control of the HSV late UL26.5promoter (Zhou et al., 2000) by means of Lipofectamine 2000 (LifeTechnologies), and then grown in SK-OV-3 cells. Virus growth wasmonitored by green fluorescence. The structure of the recombinants wasverified by sequencing the entire gB and also gD and gH ORFs forR-BP909, the scFv HER2 and the insertion site in gB of R-BP903 andR-BP901. Virus stocks were generated and titrated in SK-OV-3 cells.

To reconstitute the recombinant viruses R-BP901, R-BP903, R-313, 500 ngof recombinant BAC DNA was transfected into SK-OV-3 cells by means ofLipofectamine 2000 (Life Technologies). Virus growth was monitored bygreen fluorescence. The R-313 virus was passaged six times in SK-OV-3,frozen/thaw to lyse the SK-OV-3 cells and subsequently growth inVero-GCN4 cells. Virus stocks were generated in Vero-GCN4 and titratedin Vero-GCN4, wt-Vero and SK-OV-3 cells. The structure of therecombinant R-313 was verified by sequencing the GCN4 and the insertionsite in gB.

Example 2: Verification of Expression of the Chimeric scFv-HER2-gB ofR-BP901 and R-BP909

SK-OV-3 cells were infected at an input multiplicity of infection of 3PFU/cell with R-BP901, R-BP909, and with R-LM5, for comparison, andharvested 72 h after infection. Cell lysates were subjected topolyacrylamide gel electrophoresis, transferred to PVDF membranes andimmunoblotted with monoclonal antibody (H1817) to gB. FIG. 2 shows thatthe chimeric scFv-HER2-gB from R-BP901 and R-BP909 migrated with aslower electrophoretic mobility than wt-gB from R-LM5, and an apparentM_(r) of 130 KDaltons. Arrows point to the migration position ofchimeric and wt gB. figures to the left indicate the migration positionof molecular weight markers, expressed in kDaltons.

Example 3: Infection of J Cells Expressing Single Receptors withRecombinants R-BP903, R-BP909 and R-BP901

It has previously been shown that the insertion of scFv-HER2 in gDconfers to the recombinant virus R-LM113 the ability to enter cellsthrough the HER2 receptor. To provide evidence that the insertion ofscFV-HER2 at positions 43-44 or 81-82 of gB confers the ability to entercells through the HER2 receptor, the inventors made use of cells thatexpress HER2 as the sole receptor. The parental J cells express noreceptor for gD, hence cannot activate gD, and are not infected bywt-HSV. J-HER2 cells transgenically express HER2 as the sole receptor.As controls, the inventors included J-Nectin and J-HVEM cells, whichtransgenically express Nectin-1 or HVEM as receptors and are infected bywt-HSV. The indicated cells were infected with R-BP903, R-BP909 andR-BP901 and monitored for green fluorescence microscopy 24 h postinfection.

As shown in FIG. 3A, R-BP903 infected J-HER2 cells. The infection ofJ-Nectin, J-HVEM was not surprising, inasmuch as R-BP903 encodes awt-gD. This virus is retargeted to HER2 and retains the natural tropism.

The inventors engineered a recombinant carrying the scFv-HER2 inposition 43-44 of gB and the deletion of portions of receptors' bindingsites from gD. The two major receptors of gD are Nectin-1 and HVEM. Thebinding site of HVEM in gD maps to AA 1-32. The binding site of Nectin-1in mature gD is more widespread and includes the Ig-folded core andportions located between AA 35-38, 199-201, 214-217, 219-221. Theinventors deleted from R-BP903 mature gD the AA 6-38 region, i.e. thesame region which was previously deleted from R-LM113, a HSV retargetedto HER2 by insertion of the scFv-HER2 between AA 5 and 39 of mature gD.The deletion removes the entire HVEM binding site and some residuesimplicated in the interaction with Nectin-1, including portions locatedbetween AA 35-38. Even though a few AA implicated in the interactionwith Nectin-1 were deleted, R-LM113 was shown to be detargeted from bothNectin-1 and HVEM. The recombinant virus named R-BP909 failed to infectnot only J-HVEM cells, but also J-Nectin cells, and maintained theability to infect efficiently J-HER2 cells (FIG. 3B). R-BP909 tropism isstrikingly different from that of R-BP903 (compare FIG. 3A with FIG.3B). The inventors conclude that R-BP909 infection via theHER2-retargeted gB does not require the binding sites for HVEM and forNectin-1 in gD, and, consequently, the receptor-mediated gD activation.In summary, R-BP909 exhibits a fully redirected tropism, retargeted tothe HER2 receptor via gB and detargeted from gD receptors.

The recombinant R-BP901 which carries the scFv HER2 between AA 81 and 82of gB and has wt gD fails to infect J-HER2 cells; this virus is notretargeted to HER2 (FIG. 3C).

Example 4: Infection of HER2⁺ and HER2⁻ Cancer Cells

The SK-OV-3, BT-474, MDA-MB-453 HER2+ cancer cells, and the HER2⁻ HeLaand MDA-MB-231 cancer cells, and the HER2⁻ non-cancer HaCaT cells wereinfected at an input multiplicity of infection of 5 PFU/cell (astitrated in SK-OV-3) for 90 min at 37° C. with R-BP909 and R-BP903.Pictures were taken 24 h after infection at fluorescence microscope.R-BP909 infects the HER2-positive cancer cells and fails to infect theHER2-negative cells. R-BP903 infects cells irrespective of theexpression of HER2, in agreement with the lack of detargeting (FIG. 4).

Example 5: Characterization of R-BP909 Entry Pathways in J-HER2 andSK-OV-3

To prove that entry of R-BP909 into J-HER2 cells occurs through HER2 asthe cellular receptor, and to investigate the role of gD in the entrypathway of R-BP909 into SK-OV-3 cells, the inventors performed a seriesof blocking assays.

In addition, R-LM5, which carries a wt-gD and the other genomicmodifications present in R-BP909 and R-LM113, namely the insertion ofthe BAC sequences and the insertion of the GFP marker, was employed ascontrol. The inventors first confirmed that infection of R-BP909 occursthrough the HER2 receptor. Replicate monolayers of J-HER2 cells, orSK-OV-3 cells in 12-well plates were preincubated with trastuzumab, theMAb to HER2 from which the scFv-HER2 was derived or with non-immunemouse IgG (28 μg/ml final concentration). After 1 h at 37° C. ofpre-incubation with antibodies, the cells were infected at an inputmultiplicity of infection of 5 PFU/cell (as titrated in SK-OV-3) withR-BP909 and R-LM113 or R-LM5, as comparison. R-BP909 infection of bothcell types was almost abolished by trastuzumab, indicating that R-BP909uses HER2 as portal of entry, and does not make use of an off-targetpathway of entry. The finding that R-BP909 can make use of HER2 asreceptor provides evidence that the tropism of HSV can be modified byengineering a heterologous ligand in gB. Furthermore, the infection ofthe gB-retargeted HSV R-BP909 into J-HER2 cells can take place in cellswhich lack a gD receptor, cannot be activated by its cognate receptorsand cannot transmit the activation to gB. The inventors conclude thatinfection of R-BP909 does not necessitate a gD with functionalreceptor-binding sites. This validates the conclusion that theretargeted R-BP909 uses HER2 as the portal of entry in J-HER2 cells.

To elucidate the contribution of the essential glycoproteins, gD, gH/gLand as well as the portion of gB which was not modified by geneticengineering, virions were pre-incubated with MAbs to gD HD1 (1.5 ug/ml),MAbs to gB H126 (1:2000), MAb 52S to gH (ascites fluid 1:25) for 1 h at37° C. as indicated, and then allowed to adsorb to cells for 90 min. Inthe case of MAb HD1, the combination of HD1 plus trastuzumab (aliaherceptin) was also tested. Viral inocula were then removed, and cellswere overlaid with medium containing the indicated antibodies.

Infection was quantified by fluorescent activated cell sorter (FACS)(FIG. 5). MAb H126 to gB recognizes a linear epitope in Domain I of gB,with critical residue at Tyr₃₀₃. MAb 52S to gH recognizes a continuousepitope, independent of gL, with critical residues at Ser₅₃₆ and Ala₅₃₇.R-BP909 infection of both SK-OV-3 and J-HER2 cells was abolished by MAbH126 (1:2000) (FIG. 5A and B), indicating that a key functional domainin wt-gB was preserved in the chimera, and a role for gH/gL. MAb HD1failed to inhibit R-BP909 and R-LM113 infection, consistent withprevious findings (Gatta et al, 2015); the results support theconclusion that R-BP909 is retargeted to HER2 by means of gB, anddetargeted from Nectin1/HVEM in consequence of the AA 6-38 deletion inmature gD.

Example 6: Extent of Replication of Recombinants

The inventors compared the extent of replication of R-BP909 to that ofthe recombinants, R-LM113 and R-VG809 that are retargeted to HER2through the insertion of scFv-HER2 in gD and gH, respectively.Replication was measured in SK-OV-3 cells, which express HER2 andNectin-1/HVEM as receptors.

Cells were infected at an input multiplicity of infection of 0.1PFU/cell for 90 min at 37° C.; unabsorbed virus was inactivated by meansof an acidic wash (40 mM citric acid, 10 mM KCl, 135 mM NaCl [pH 3]).Replicate cultures were frozen at the indicated times (3, 24 and 48 h)after infection and the progeny was titrated in SK-OV-3. The results inFIG. 6 show that R-BP909 replicated to a similar extent to R-VG809,about one log less than R-LM113.

Example 7: Ability of R-BP909, and of R-VG809 for Comparison, to KillHER2-Positive Cancer Cells, and Lack of Killing Ability for HER2⁻ CancerCells

The HER2-positive SK-OV-3 and MDA-MB-453 and the HER2-negativeMDA-MB-231 cells were seeded in 96 well plates 8×10E3 cells/well, andexposed to the recombinant R-BP909, R-VG809 for comparison ormock-infected for 90 min at 37° C. The input multiplicity of infection(as titrated in the correspondent cell line) was 2 PFU/cell for theSK-OV-3 and MDA-MB-453 and of 0.1 PFU/cells for the MDA-231 cells.Alamar-Blue (10 μl/well Life Technologies) was added to the culturemedia at the indicated times after virus exposure and incubated for 4 hat 37° C. Plates were read at 560 and 600 nm with GloMax Discover System(Promega). For each time point, cell viability was expressed as thepercentage of AlamarBlue reduction in infected versus uninfected cells,excluding for each samples the contribution of medium alone.Cytotoxicity caused by R-BP909 and R-VG809 in HER2-positive SK-OV-3 andMDA-MB-453 ranged from 70 to 90% at 7 days after infection. Both virusesfailed to kill the HER2-negative MDA-MD-231 cancer cells, consistentwith their inability to infect these cells (FIG. 7).

Example 8 Ability of R-313 to Replicate in Vero-GCN4 and in the CancerCell Line SK-OV-3

It has previously been shown that the insertion of scFv-HER2 in place ofAA 6-38 of gD confers to the recombinant virus R-LM113 the retargetingto HER2 receptor and the detargeting from both Nectin-1 and HVEM. In thepresent invention the inventors provide evidence that R-BP909, whichcarries the scFv-HER2 between AA 43-44 of gB, exhibits a fullyredirected tropism, retargeted to the HER2 receptor via gB.

The inventors further investigated whether gB is a suitable glycoproteinin order to retarget HSV by means of a short peptide, exemplified hereby the epitope YHLENEVARLKK (SEQ ID NO: 38) of GCN4 yeast transcriptionfactor with two flanking wt GCN4 residues and two GS linkers, hereinnamed GCN4 peptide. The 20 amino acid peptide should confer to R-313 theability to infect and replicate in the Vero-GCN4 cell line, expressingthe artificial receptor made of the scFv to GCN4 (Zahnd et al., 2004)fused to extracellular domains 2, 3, TM and C-tail of Nectin-1.

To test the tropism of the R-313 the inventors made use of simianwt-Vero, Vero-GCN4, SK-OV-3 and of the previously described J cellsexpressing or not receptors for gD. The indicated cells were infectedwith R-313 and, where indicated, the cells were pretreated withTrastuzumab (alias Herceptin) (28 μg/ml final concentration). Theinfection was monitored by green fluorescence microscopy 24 h afterinfection.

As shown in FIG. 8, R-313 infected both untreated Vero-GCN4 and Vero-wt,but in the presence of Trastuzumab, only the infection in Vero-GCN4 wasobserved. This result indicates that R-313 was able to infect Vero-GCN4.In contrast to the infection with R-313 of wt-Vero cells, this infectionwas not inhibited by herceptin, indicating that it was indeed mediatedby the GCN4 peptide inserted in gB. The infection with R-313 of wt-Verocells occurs through the simian ortholog of HER2 present in Vero cells,as it is indeed inhibited by exposure of cells to herceptin.

The scFv-HER2 fused to gD still enabled infection of SK-OV-3 cellsthrough HER2, as documented by inhibition by herceptin. The lack ofinfection of J, J-Nectin and J-HVEM confirmed the deretargeting profilealready exhibited by R-LM113, due to the deletion of AA 6-38 of gD.Cumulatively this series of results indicates that R-313 has the abilityto infect Vero-GCN4 cells through the GCN4 peptide fused in gB, and theSK-OV-3 cells through HER2 in gD.

Example 9: Extent of Replication of R-313 in Vero-GCN4 and in SK-OV-3Cells

The inventors compared the extent of replication of R-313 to that of therecombinants R-LM113 and R-LM5 in Vero-GCN4 and in SK-OV-3 cells. Cellswere infected at an input multiplicity of infection of 0.1 PFU/cell (astitrated in the correspondent cell line) for 90 min at 37° C.;unabsorbed virus was inactivated by means of an acidic wash (40 mMcitric acid, 10 mM KCl, 135 mM NaCl [pH 3]). Replicate cultures werefrozen at the indicated times (3, 24 and 48 h) after infection and theprogeny was titrated in SK-OV-3. The results in FIG. 9 show that R-313replicated in Vero-GCN4 to a higher extent than R-LM113, and to similarextent as R-LM5. R-313 can replicate in SK-OV-3 to a similar extent asR-LM113, and almost one log lower than R-LM5.

Cumulatively, the results show that R-313 is simultaneously retargetedthrough GCN4 and through HER2.

Example 10: Plating Efficiency of R-313 in Different Cell Lines

For plating efficiency experiments, the indicated cell monolayers wereinfected with replicate aliquots of serial dilutions (from 10⁻⁵ to10⁻¹⁰) of R-313. After infection and removal of inoculum, mediumcontaining agar was added to the plates and monolayers were incubatedfor 3 days at 37° C. to allow plaque formation. At 3^(th) day plaqueswere scored under the fluorescence microscope. Figures indicate that theR-313 plating efficiency in SK-OV-3 is very similar to that inVero-GCN4; both are slightly higher than that observed in wt-Vero cells,confirming that R-313 can make use alternatively of the GCN4 peptideengineered in gB and of the scFv-HER2 inserted in gD to enter Vero-GCN4and SK-OV-3 cells, respectively. The plating efficiency of R-313 inJ-HER2 cells could not be differentiated from that R-LM113 in the samecells, indicating that the insertion of the GCN4 peptide is notdetrimental (FIG. 10).

Example 11: Relative Plaque Size of R-313 in Different Cell Lines

To perform a plaque size assay, 10-fold dilutions of R-313, R-LM113 andR-LM5 were plated onto Vero-GCN4, wt-Vero and SK-OV-3 monolayers. Theinfected monolayers were overlaid with medium containing agar. Threedays later pictures were taken at the fluorescence microscope.Representative pictures show that in any cell line tested R-313 formslarger plaques than R-LM113. In turn, plaques formed by R-LM5 were evenlarger that those formed by R-313 (FIG. 11).

Example 12

A) R-315: insertion of GCN4 peptide between AA 81 and 82 of HSV gB inHSV recombinant already expressing a scFv-HER2 in the deletion of AA6-38 in gD.

The procedure was the same as described above to engineer the GCN4peptide in gB of R-313, with the following differences. First, the galKcassette was amplified by means of primers gB81fGALK

CGGGGGACACGAAACCGAAGAAGAACAAAAAACCGAAAAACCCACCGCCGCCGCCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 15) and gB81GALKrevCGCAGGGTGGCGTGGCCCGCGGCGACGGTCGCGTTGTCGCCGGCGGGGC GTCAGCACTGTCCTGCTCCTT(SEQ ID NO: 16) using pGalK as template. Next, the GCN4 peptide cassette(SEQ ID NO: 36, encoding SEQ ID NO: 37) with the downstream and upstreamSer-Gly linkers and bracketed by homology arms to gB was generatedthrough the annealing and extension of primers gB_81_GCN4_forCGGGGGACACGAAACCGAAGAAGAACAAAAAACCGAAAAACCCACCGCCGCCGGGATCCAAGAACTACCACCTGGAGAACGAGGTGGCCAGACTGAAGAAGC TGGTGGGCAGC (SEQ IDNO: 49) and gB_81_GCN4_revCGCAGGGTGGCGTGGCCCGCGGCGACGGTCGCGTTGTCGCCGGCGGGGCGGCTGCCCACCAGCTTCTTCAGTCTGGCCACCTCGTTCTCCAGGTGGTAGTT CTTGGATCC (SEQ IDNO: 50) which introduce a silent restriction site for the BamHIendonuclease, useful for screening of colonies by means of restrictionanalysis. The recombinant genome encodes the chimeric gB (SEQ ID NO:51), which carries the GCN4 peptide including one downstream and oneupstream Ser-Gly linker with the sequence GS.

B) R-317: insertion of GCN4 peptide between AA 76 and 77 of HSV gB inHSV recombinant already expressing a scFv-HER2 in the deletion of AA6-38 in gD.

The procedure was the same as described above to engineer the GCN4peptide in gB of R-315, with the following differences. First, the galKcassette was amplified by means of primers gB_76_galK_forGGCCCCGCCCCAACGGGGGACACGAAACCGAAGAAGAACAAAAAACCGAAACCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 44) and gB_76_galK_revCCCGCGGCGACGGTCGCGTTGTCGCCGGCGGGGCGCGGCGGCGGTGGGT TTCAGCACTGTCCTGCTCCTT(SEQ ID NO: 45) using pGalK as template. Next, the GCN4 peptide cassette(SEQ ID NO: 36, encoding SEQ ID NO: 37) with the downstream and upstreamSer-Gly linkers and bracketed by homology arms to gB was generatedthrough the annealing and extension of primers gB_76_GCN4_forGGCCCCGCCCCAACGGGGGACACGAAACCGAAGAAGAACAAAAAACCGAAAGGATCCAAGAACTACCACCTGGAGAACGAGGTGGCCAGACTGAAGAAGCTG GTGGGCAGC (SEQ IDNO: 46) and gB_76_GCN4_revCCCGCGGCGACGGTCGCGTTGTCGCCGGCGGGGCGCGGCGGCGGTGGGTTGCTGCCCACCAGCTTCTTCAGTCTGGCCACCTCGTTCTCCAGGTGGTAGTT CTTGGATCC (SEQ IDNO: 47) which introduce a silent restriction site for the BamHIendonuclease, useful for screening of colonies by means of restrictionanalysis. The recombinant genome encodes the chimeric gB (SEQ ID NO:48), which carries the GCN4 peptide including one downstream and oneupstream Ser-Gly linker with the sequence GS.

C) R-319: insertion of GCN4 peptide between AA 95 and 96 of HSV gB inHSV recombinant already expressing a scFv-HER2 in the deletion of AA6-38 in gD. The procedure was the same as described above to engineerthe GCN4 peptide in gB of R-317, with the following differences. First,the galK cassette was amplified by means of primers gB_95_galK_forCGCCGCCGCGCCCCGCCGGCGACAACGCGACCGTCGCCGCGGGCCACGCCCCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 52) and gB_95_galK_revGTTTGCATCGGTGTTCTCCGCCTTGATGTCCCGCAGGTGCTCGCGCAGGGTT CAGCACTGTCCTGCTCCTT(SEQ ID NO: 53) using pGalK as template. Next, the GCN4 peptide cassette(SEQ ID NO: 36, encoding SEQ ID NO: 37) with the downstream and upstreamSer-Gly linkers and bracketed by homology arms to gB was generatedthrough the annealing and extension of primers gB_95_GCN4_forCGCCGCCGCGCCCCGCCGGCGACAACGCGACCGTCGCCGCGGGCCACGCCGGATCCAAGAACTACCACCTGGAGAACGAGGTGGCCAGACTGAAGAAGCT GGTGGGCAGC (SEQ IDNO: 54) and gB_95_GCN4_revGTTTGCATCGGTGTTCTCCGCCTTGATGTCCCGCAGGTGCTCGCGCAGGGTGCTGCCCACCAGCTTCTTCAGTCTGGCCACCTCGTTCTCCAGGTGGTAGTTC TTGGATCC (SEQ IDNO: 55) which introduce a silent restriction site for the BamHIendonuclease, useful for screening of colonies by means of restrictionanalysis. The recombinant genome encodes the chimeric gB (SEQ ID NO:56), which carries the GCN4 peptide including one downstream and oneupstream Ser-Gly linker with the sequence GS.

To reconstitute the recombinant virus R-BP909, 500 ng of recombinant BACDNA was transfected into the gD-complementing cell line named R6 (rabbitskin cell line expressing wt-gD under the control of the HSV late UL26.5promoter (Zhou et al., 2000) by means of Lipofectamine 2000 (LifeTechnologies), and then grown in SK-OV-3 cells. Virus growth wasmonitored by green fluorescence. The structure of the recombinants wasverified by sequencing the entire gB and also gD and gH ORFs forR-BP909, the scFv HER2 and the insertion site in gB of R-BP903 andR-BP901. We identified for gB of the recombinant virus R-909 onemutation (Y276S), not present in the engineered BAC-DNA. Virus stockswere generated and titrated in SK-OV-3 cells.

To reconstitute the recombinant viruses R-BP901, R-BP903, R-313, R-315,R-317 and R-319 500 ng of recombinant BAC DNA was transfected intoSK-OV-3 cells by means of Lipofectamine 2000 (Life Technologies). Virusgrowth was monitored by green fluorescence. The R-313 virus was passagedsix times in SK-OV-3, frozen/thawed to lyse the SK-OV-3 cells andsubsequently growth in Vero-GCN4R cells. Virus stocks were generated inVero-GCN4R and titrated in Vero-GCN4R, wt-Vero and SK-OV-3 cells. Thegenome of the recombinant R-313, R-315, R-317 and R-319 was partiallyverified by sequencing the entire gB.

Example 13: Ability of R-313, R-315, R-317 and R-319 to Replicate inVero-GCN4R and in the Cancer Cell Line SK-OV-3

It has previously been shown that the insertion of scFv-HER2 in place ofAA 6-38 of gD confers to the recombinant virus R-LM113 the retargetingto HER2 receptor and the detargeting from both Nectin-1 and HVEM. In thepresent invention the inventors provide evidence that R-BP909, whichcarries the scFv-HER2 between AA 43-44 of gB, exhibits a fullyredirected tropism, retargeted to the HER2 receptor via gB.

The inventors further investigated whether gB is a suitable glycoproteinin order to retarget HSV by means of a short peptide, exemplified hereby the epitope YHLENEVARLKK (SEQ ID NO: 38) of GCN4 yeast transcriptionfactor with two flanking wt GCN4 residues and two GS linkers, hereinnamed GCN4 peptide. The 20 amino acid peptide should confer to R-313,R-315, R-317 and R-319 the ability to infect and replicate in theVero-GCN4R cell line, expressing the artificial receptor made of thescFv to GCN4 (Zahnd et al., 2004) fused to extracellualr domains 2, 3,TM and C-tail of Nectin-1.

To test the tropism of the R-313, R-315, R-317 and R-319, the inventorsmade use of simian wt-Vero, Vero-GCN4R, SK-OV-3 and of the previouslydescribed J cells expressing or not receptors for gD. The indicatedcells were infected with the indicated recombinant and, where indicated,the cells were pretreated with Trastuzumab (alias Herceptin) (28 μg/mlfinal concentration). The infection was monitored by green fluorescencemicroscopy 24 h after infection.

As shown in FIG. 8, R-313 (panel A), R-315 (panel B), R-317 (panel C)and R-319 (panel D) infected both untreated Vero-GCN4R and Vero-wt, butin the presence of Trastuzumab (alias Herceptin), only the infection inVero-GCN4R was observed. This result indicates that all the recombinantviruses that carry the insertion of GCN4 peptide in different positionof gB were able to infect Vero-GCN4R. In contrast to the infection ofwt-Vero cells, the infection of Vero-GCN4R, was not inhibited byHerceptin, indicating that it was indeed mediated by the GCN4 peptideinserted in gB. The infection with R-313,R-315, R-317 and R-319 ofwt-Vero cells occurs through the simian ortholog of HER2 present in Verocells, as it is indeed inhibited by exposure of cells to Herceptin.

The scFv-HER2 inserted in gD still enabled infection of SK-OV-3 cellsthrough HER2, as documented by inhibition by Herceptin. The lack ofinfection of J, J-Nectin and J-HVEM confirmed the deretargeting profilealready exhibited by R-LM113, due to the deletion of AA 6-38 of gD.Cumulatively this series of results indicates that R-313, R-315, R-317and R-319 have the ability to infect Vero-GCN4R cells through the GCN4peptide inserted in gB, and the SK-OV-3 cells through HER2 in gD.

Example 14: Extent of Replication of R-313, R-315, R-317 and R-319 inVero-GCN4R and in SK-OV-3 Cells

The inventors compared the extent of replication of R-313, R-315, R-317,R-319 to that of the recombinants R-LM113 and R-LM5 in SK-OV-3 cells(FIG. 9 A) and in Vero-GCN4R (FIG. 9 B). Cells were infected at an inputmultiplicity of infection of 0.1 PFU/cell (as titrated in thecorrespondent cell line) for 90 min at 37° C.; unabsorbed virus wasinactivated by means of an acidic wash (40 mM citric acid, 10 mM KCl,135 mM NaCl [pH 3]). Replicate cultures were frozen at the indicatedtimes (24 and 48 h) after infection and the progeny was titrated inSK-OV-3. It can be seen from FIG. 9 A that R-315 and R-317 grew tosimilar titers as R-LM5 and R-LM113 in SK-OV-3 cells. In contrast, R-313and R-319 grew about one-two log less than R-315 and R-317. The resultsin FIG. 9 B show that R-313, R-315 and R-317 replicated in Vero-GCN4R toa similar extent as R-LM113, and one log lower than R-LM5. In turn,R-319 grew about one-two log less than R-315, R-317 and R-319.

Cumulatively, the results show that R-313, R-315, R-317 and R-319 aresimultaneously retargeted through GCN4 and through HER2.

Example 15: Plating Efficiency of R-313, R-315, R-317 and R-319 inDifferent Cell Lines

The inventors compared the ability of R-313, R-315, R-317 and R-319 toform plaques in different cell lines, with respect to the number (FIG.10). Replicate aliquots of R-LM5, R-LM113, R-313, R-315, R-317 andR-319, containing a same amount of virus (50 PFU), as titrated inSK-OV-3 cells, were plated on wt-Vero, Vero-GCN4R and SK-OV-3. Theinfected monolayers were overlaid with medium containing agar and thenumber of plaques was scored 3 days later.

The results of the experiment indicate that the plating efficiency ofall recombinant virus carrying the GCN4 peptide insertion in gB (R-313,R-315, R-317 and R-319), but not of the control viruses, was higher onVero-GCN4R cells in comparison to wt-Vero. All the gB-recombinantsexhibited similar plating efficiency in Vero-GCN4R and in SK-OV-3 cells.

Example 16: Relative Plaque Size of R-313, R-315, R-317 and R-319 inDifferent Cell Lines

To perform a plaque size assay, 10-fold dilutions of R-313, R-315, R-317and R-319 were plated onto Vero-GCN4R, wt-Vero and SK-OV-3 monolayers.The infected monolayers were overlaid with medium containing agar. Threedays later pictures were taken at the fluorescence microscope.Representative pictures show that in any cell line tested R-313, R-315,R-317 and R-319 form larger plaques than R-LM113. Plaques formed byR-LM5 were even larger (FIG. 11). For plaque size determinations (FIG.11 B), pictures of 5 plaques were taken for each virus. Plaque areas(pxE2) were measured with Nis Elements-Imaging Software (Nikon). Eachresult represents average areas±SD.

Example 17: R-321: Reintroduction of AA 6-29 and 31-37 of gD in HSVRecombinant Already Expressing a scFv-HER2 in the Deletion of AA 6-38 ingD and GCN4 Peptide Between AA 43 and 44 of gB

First, the galK cassette was amplified by means of primers gD5_galK_f

TTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGCCTGTTGACAATTAATCATCGGCA (SEQ ID NO: 57) and scFv_galK_revGAGGCGGACAGGGAGCTCGGGGACTGGGTCATCTGGATATCGGAATTCTCT CAGCACTGTCCTGCTCCTT(SEQ ID NO: 58) using pGalK as template. The galK cassette was insertedin R-313 backbone by means of galK recombineering. Next, the oligo thatcomprises AA 6-29 and 31-37 of gD was generated through the annealingand extension of primers gDdel30_38 forTTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGGATGCCTCTCTCAAGATGGCCGACCCCAATCGCTTTCGCGGCAAAGACCTTCC GGTCC (SEQ ID NO:59) and gDde130_38 revGAGGCGGACAGGGAGCTCGGGGACTGGGTCATCTGGATATCGGAATTCTCCACGCGCCGGACCCCCGGAGGGGTCAGCTGGTCCAGGACCGGAAGGTCTTT GCCGCGA (SEQ ID NO:60). The recombinant genome encodes the chimeric gD (SEQ ID NO: 61),which carries the deletion of AA 30 and 38 of gD and the insertion ofscFv-HER2 after AA 37 of gD. SEQ ID NO: 35 shows the chimeric gB havinginserted the GCN4 peptide between amino acids 43 and 44. The structureof the recombinant BAC was verified by sequencing the upstream anddownstream the region 6-37 of gD.

To reconstitute the recombinant virus R-321, 500 ng of recombinant BACDNA was transfected into SK-OV-3 cells by means of Lipofectamine 2000(Life Technologies). Virus growth was monitored by green fluorescence.The R-321 virus was passaged six times in SK-OV-3, frozen/thaw to lysethe SK-OV-3 cells and subsequently growth in Vero-GCN4R cells.

Example 18: R-321 is Retargeted from HSV-1 Natural Receptors

It has previously been shown that the insertion of scFv-HER2 in place ofAA 6-38 of gD confers to the recombinant virus R-LM113 the retargetingto HER2 receptor and the detargeting from both Nectin-1 and HVEM. In thepresent invention the inventors provide evidence that R-321, whichcarries the deletion of only AA 30 and 38 of gD and the insertion ofscFv-HER2 after AA 37 of gD, exhibits a fully de-targeted profile, sinceit loss the ability to infect trough HSV-1 natural receptors. Moreover,R-321 carries the GCN4 peptide between AA 43 and 44 of gB, like R-313.

To test the tropism of the R-321, inventors made use of simian wt-Vero,Vero-GCN4R, SK-OV-3 and of the previously described J cells expressingor not receptors for gD. The indicated cells were infected with R-321and, where indicated, the cells were pretreated with Trastuzumab (aliasHerceptin) (28 μg/ml final concentration). The infection was monitoredby green fluorescence microscopy 24 h after infection.

The lack of infection of J, J-Nectin and J-HVEM (FIG. 15) indicates thatR-321 is de-targeted from HSV-1 natural receptors, due to the deletionof AA 30 and 38 of gD. The scFv-HER2 fused to gD enabled infection ofSK-OV-3 cells through HER2, as documented by inhibition by Herceptin. Asshown in FIG. 15, R-321 infected both untreated Vero-GCN4R and Vero-wt,but in the presence of Trastuzumab (alias Herceptin), only the infectionin Vero-GCN4R was observed. This result indicates that R-321 is able toinfect Vero-GCN4R, as R-313. In contrast to the infection of wt-Verocells, the infection of Vero-GCN4R, was not inhibited by Herceptin,indicating that it was indeed mediated by the GCN4 peptide inserted ingB. The infection with R-321 occurs through the simian ortholog of HER2present in Vero cells, as it is indeed inhibited by exposure of cells toHerceptin.

Cumulatively, the results show that R-321 is simultaneously retargetedthrough GCN4 and through HER2 and de-targeted from HSV natural receptoras a consequence of deletion of aa 30 and 38 in gD.

Example 19: Extent of Replication of R-321 in Vero-GCN4R and in SK-OV-3Cells

The inventors compared the extent of replication of R-321 to that of therecombinants R-LM113 and R-LM5 in SK-OV-3 cells (FIG. 16 A) and inVero-GCN4R (FIG. 16 B). Cells were infected at an input multiplicity ofinfection of 0.1 PFU/cell (as titrated in the correspondent cell line)for 90 min at 37° C.; unabsorbed virus was inactivated by means of anacidic wash (40 mM citric acid, 10 mM KCl, 135 mM NaCl [pH 3]).Replicate cultures were frozen at the indicated times (24 and 48 h)after infection and the progeny was titrated in SK-OV-3. It can be seenfrom FIG. 16 A that R-321 grew to similar titers as R-LM5 and R-LM113 inSK-OV-3 cells. The results in FIG. 16 B show that R-321 replicated inVero-GCN4R one log higher than R-LM113, and to similar extent thanR-LM5.

Example 20: Vero-GCN4 Cell Line

The Vero GCN4 cell line expresses an artificial chimeric receptor, madeof an scFv to the GCN4 peptide (Zahnd et al., 2004), with the sequenceoptimized for human codon usage as reported in SEQ ID NO: 39, fused toNectin-1. The GCN4 peptide is part of the Saccharomyces cerevisiaetranscription factor GCN4, whose partial mRNA sequence is reported inSEQ ID NO 42. More in detail, an N-terminal leader peptide and HA tagsequence is present like in the pDISPLAY (Invitrogen) vector. Thisshould ensure efficient and proper processing of the leader peptide.After the HA tag, a short GA linker is present upstream of the scFv. Theamino acid sequence of the scFv to GCN4 is reported in SEQ ID NO: 39.C-terminal to the scFv a short GSGA linker is present. The rest of themolecule corresponds to human Nectin-1 (PVRL1) residues Met143 to Val517comprising the Nectin-1 extracellular domains 2 and 3, the TM segmentand the intracellular cytoplasmic tail (FIG. 12). The chimera wassynthesized in vitro by Gene Art, and cloned into pcDNA3.1—Hygro_(+),resulting in plasmid scFv_GCN4_Nectin1 chimera, whose insert has thenucleotide sequence identified by SEQ ID NO: 40, encoding the amino acidsequence of the scFv-GCN4 nectin1 chimera SEQ ID NO: 41.

The DNA from plasmid scFv_GCN4_Nectin1 chimera was transfected into Verocells (ATCC CCL-81™) by means of Lipofectamine 2000. Vero cellsexpressing the artificial receptor to the GCN4 peptide were selected bymeans of Hygromycin (200 μg/ml), and subsequently sorted by means ofmagnetic beads (Miltenyi), in combination with MAb to HA tag. The sortedcells were subjected to single cell cloning in 96 well (0.5 cell/well).

Single clones were analysed by FACS for detection of expression of thescFv to the GCN4 peptide by means of MAb to HA tag. The selected clonewas 11.2. We ascertained that during serial passages of the Vero-GCN4cell line, the expression of the artificial receptor remained stableafter 40 consecutive passages (FIG. 13).

REFERENCES

-   Abstract # P-28, 9^(th) International conference on Oncolytic virus    Therapeutics, Boston 2015-   Arndt K. and Fin G. R., PNAS 1986, 83, 8516-8520-   Backovic M. et al., PNAS, 2009, 106, 2880-2885;-   Burke H. G. and Heldwein E. E., Plos Pathogens, 2015, 11(11),    e1005300, doi: 10.1371/journal.ppat.1005300-   Castoldi R. et al., Oncogene, 2013, 32, 5593-601-   Castoldi R. et al., Protein Eng Des Sel, 2012, 25, 551-9-   Douglas J. T. et al., Nat Biotechnol, 1999, 17, 470-475-   Florence G. et al., Virology: A Laboratory Manual, 1992, ISBN-13:    978-0121447304-   Gallagher J. R. et al., PLOS Pathogens, 2014, 10, e1004373, 1-16-   Gatta V. et al., PLOS Pathogens, 2015, DOI:    10.1371/journal.ppat.1004907-   Heldwein E. E et al., Science, 2006, 313, 217-220-   Hope I. A and Struhl K., EMBO J, 1987, 6, 2781-2784-   Josan J. S. et al., Bioconjug Chem, 2011, 22, 1270-1278;-   Karlin S. and Altschul S. F., PNAS, 1990, 87, 2264-2268-   Karlin S. and Altschul S. F., PNAS, 1993, 90, 5873-5877-   Lin E. and Spear P. G., PNAS, 2007, 104, 13140-13145-   Liu B. L. et al., Gene Ther, 2003, 10, 292-303-   Morgan A. A. and Rubistein E., PLoS One, 2013, 8(1), e53785. doi:    10.1371/journal.pone.0053785. Epub 2013 Jan. 25. PHD: 23372670-   Menotti L, et al., J Virol, 2008, 82, 10153-10161; doi:    10.1128/JVI.01133-08. Epub 2008 Aug. 6.-   Nakamura T. et al., Nat Biotechnol, 2005, 23, 209-214. Epub 2005    Jan. 30-   Needleman S. B. and Wunsch C. D., J Mol Biol, 1970, 48, 443-453-   Pearson W. R. and Lipman D. J., PNAS, 1988, 85, 2444-2448-   Peterson R. B. and Goyal S. M., Comp Immunol Microbiol Infect Dis.    1988, 11, 93-98-   Potel C. et al., Journal of Virological Methods, 2002, 105, 13-23-   Sandri-Goldin R. M. et al., Alpha Herpesviruses: Molecular and    Cellular Biology,-   Caister Academic Press, 2006-   Shallal H. M. et al., Bioconjug Chem, 2014, 25, 393-405-   Smith T. F. and Waterman M. S., Add APL Math, 1981, 2, 482-489-   Xu L. et al., PNAS, 2012, 109, 21295-21300-   Zahnd C. et al., J Biol Chem 2004; 279, 18870-18877-   Zhou, G. et al., J Virol, 2000, 74, 11782-11791

1. A recombinant herpesvirus comprising a heterologous polypeptideligand capable of binding to a target molecule and fused to or insertedinto glycoprotein B (gB) present in the envelope of the herpesvirus,wherein the ligand is fused to gB, or wherein the ligand is inserted atany amino acid within a disordered region of gB, but is not inserted atany amino acid within the region spanning from amino acids 77 to 88 ofgB according to SEQ ID NO: 1 or within a corresponding region of ahomologous gB, or wherein the ligand is inserted at any amino acidwithin a region spanning from amino acids 31 to 77 or 88 to 184,preferably amino acids 31 to 77 or 88 to 136 or more preferably 31 to 77or 88 to 108, and/or within a region spanning from amino acids 409 to545, preferably amino acids 459 to 545, more preferably amino acids 459to 497, or still more preferably amino acid 460 to 491, of gB accordingto SEQ ID NO: 1 or within a corresponding region of a homologous gB, orwherein the ligand has a length of 5 to 120 amino acids and is insertedat any amino acid within a region spanning from amino acids 77 to 88 ofgB according to SEQ ID NO: 1 or within a corresponding region of ahomologous gB.
 2. (canceled)
 3. The herpesvirus of claim 1, wherein theherpesvirus has the capability of binding to a cell expressing orbinding the target molecule, preferably of fusing with the cellmembrane, more preferably of entering the cell, most preferably ofkilling the cell.
 4. The herpesvirus according to claim 1, wherein thetarget molecule is present on a diseased cell, preferably wherein thediseased cell is a tumor cell, an infected cell, a degenerativedisorder-associated cell or a senescent cell, or wherein the targetmolecule is present on a cell present in cell culture, preferablywherein the cell is a cultured cell suitable for growth of theherpesvirus, more preferably a cell line approved for herpesvirusgrowth, even more preferably a Vero, 293, 293T, HEp-2, HeLa, BHK, or RScell, most preferably a Vero cell.
 5. The herpesvirus according to claim1, wherein the target molecule present on a diseased cell is atumor-associated receptor, preferably a member of the EGF receptorfamily, including HER2, EGFR, EGFRIII, or EGFR3 (ERBB3), EGFRvIII, orMET, FAP, PSMA, CXCR4, CEA, CADC, Mucins, Folate-binding protein, GD2,VEGF receptors 1 and 2, CD20, CD30, CD33, CD52, CD55, the integrinfamily, IGF1R, the Ephrin receptor family, the protein-tyrosine kinase(TK) family, RANKL, TRAILR1, TRAILR2, IL13Ralpha, UPAR, Tenascin, amember of the immune checkpoint family regulators, including PD-1,PD-L1, CTL-A4, TIM-3, LAG3, or DO, tumor-associated glycoprotein 72,ganglioside GM2, A33, Lewis Y antigen, or MUC1, most preferably HER2, orwherein the target molecule present on a cell present in cell culture isan artificial molecule, preferably an antibody, an antibody derivativeor an antibody mimetic, more preferably a single-chain antibody (scFv),still more preferably an scFv capable of binding to a part of the GCN4yeast transcription factor, still more preferably an scFv capable ofbinding to the part of the GCN4 yeast transcription factor as comprisedby SEQ ID NO: 37, still more preferably the scFv as comprised by SEQ IDNO: 39, most preferably the molecule identified by the sequence of SEQID NO:
 41. 6. The herpesvirus according to claim 1, wherein the ligandis a natural polypeptide or an artificial polypeptide, preferablywherein the ligand is capable of binding to a target molecule present ona cell present in cell culture or to a target molecule present on adiseased cell, more preferably wherein the ligand is a natural ligand ofa target molecule which is accessible on a cell, a part of the naturalligand capable of binding to the target molecule, a part of a naturalpolypeptide, an antibody, an antibody derivative, an antibody mimetic,more preferably wherein the ligand is a part of the natural polypeptidecapable of binding to a target molecule present on a cell present incell culture or an scFv, still more preferably wherein the ligand is apart of the GCN4 yeast transcription factor such as the part of the GCN4yeast transcription factor as comprised by SEQ ID NO: 37 or an scFvcapable of binding to a target molecule present on a tumor cell,preferably HER2, most preferably wherein the ligand is the moleculeidentified by the sequence of SEQ ID NO: 37 or the scFv identified bySEQ ID NO:
 32. 7. The herpesvirus according to claim 4, wherein thetarget molecule is HER2, the ligand is an scFv as identified by SEQ IDNO: 32 and the diseased cell is a tumor cell expressing HER2, preferablya breast cancer cell, ovary cancer cell, stomach cancer cell, lungcancer cell, head and neck cancer cell, osteosarcoma cell, glioblastomamultiforme cell, or salivary gland tumor cell, and/or wherein the targetmolecule is the molecule with the sequence of SEQ ID NO: 41, the ligandis the molecule identified by the sequence of SEQ ID NO: 37, and thecell is present in cell culture and expresses the molecule identified bythe sequence of SEQ ID NO:
 41. 8. The herpesvirus according to claim 1,wherein one or more ligands are fused to or inserted into gB, preferablywherein the gB comprises a ligand capable of binding to a targetmolecule present on a cell present in cell culture and a ligand capableof binding to a target molecule present on a diseased cell.
 9. Theherpesvirus according to claim 1, wherein the herpesvirus comprises amodified gD and/or a modified gH, preferably wherein the gB comprises aligand capable of binding to a target molecule present on a cell presentin cell culture and the modified gD and/or the modified gH comprise(s) aligand capable of binding to a target molecule present on a diseasedcell, most preferably wherein the gB comprises the sequence identifiedby SEQ ID NO: 37, the target molecule is the molecule with the sequenceidentified by SEQ ID NO: 41, and the cell is present in cell culture andexpresses the molecule identified by the sequence of SEQ ID NO: 41, andthe modified gD and/or the modified gH comprise(s) an scFv identified bySEQ ID NO: 32, the target molecule is HER2, and the cell is a tumor cellexpressing HER2, preferably a breast cancer cell, ovary cancer cell,stomach cancer cell, lung cancer cell, head and neck cancer cell,osteosarcoma cell, glioblastoma multiforme cell, or salivary gland tumorcell.
 10. The herpesvirus according to claim 9, wherein the gD ismodified to have a deletion of amino acids 30 to 38 of gD or a subsetthereof, preferably wherein the gD is modified to have a deletion ofamino acid 30 and/or amino acid 38, more preferably wherein the gD ismodified to have a deletion of amino acid 30 and amino acid 38, withregard to mature gD according to SEQ ID NO: 62 or a corresponding regionof a homologous gD.
 11. The herpesvirus according to claim 10, wherein aheterologous polypeptide ligand is inserted into gD instead of aminoacids 30 to 38 or a subset thereof, preferably wherein the heterologouspolypeptide ligand is inserted instead of amino acid 30 or amino acid38, more preferably wherein the heterologous polypeptide ligand isinserted instead of amino acid 38 and amino acid 30 is deleted, withregard to mature gD according to SEQ ID NO: 62 or a corresponding regionof a homologous gD.
 12. The herpesvirus according to claim 1, whereinthe herpesvirus encodes one or more molecules that stimulate(s) the hostimmune response against a cell, preferably a diseased cell.
 13. Apharmaceutical composition comprising the herpesvirus according to claim1 and a pharmaceutically acceptable carrier, optionally additionallycomprising one or more molecule(s) that stimulate(s) the host immuneresponse against a cell, preferably a diseased cell.
 14. (canceled) 15.A nucleic acid molecule comprising a nucleic acid coding for the gB ofthe herpesvirus according to claim 1, having fused or inserted theligand, or a vector comprising said nucleic acid molecule, or apolypeptide comprising said gB, having fused or inserted the ligand, ora cell comprising said herpesvirus, said nucleic acid molecule, saidvector, or said polypeptide. 16-21. (canceled)